Cryptococcus neoformans and also discuss other major melanotic pathogenic fungi. Melanin interacts with the immune system through diverse pathways, reducing the effectiveness of phagocytic cells, binding effector molecules and antifungals, and modifying complement and antibody responses.Melanins are ubiquitous complex polymers that are commonly known in humans to cause pigmentation of our skin. Melanins are also present in bacteria, fungi, and helminths. In this review, we will describe the diverse interactions of fungal melanin with the mammalian immune system. We will particularly focus on  Melanins are a family of structurally complex dark pigment polymer present in all biological kingdoms . The poll-3,4-dihyroxyphenylalanine (L-DOPA)  and (L-DOPA) . In the The L-DOPA pathway is similar to mammalian melanin biosynthesis, where the pathway typically uses either L-DOPA or tyrosine as starting molecules. If the pathway starts with tyrosine, tyrosinase will perform a two-step oxidation, turning tyrosine into dopaquinone. Similarly, laccase is the enzyme responsible for converting L-DOPA into dopaquinone. Dopaquinone is then turned into leucodopachrome (cyclodopa) and then oxidized to dopachrome. Dopachrome then goes through tautomerization to form dihydroxyindoles, which are simultaneously oxidized and polymerized to produce DOPA melanin .C. neoformans is one of the most well studied pathogens for melanization  via UV irradiation and observed that the mutants lacked virulence as the cells were cleared from mouse organs, whereas wild-type (WT) cells expanded in numbers, especially in the brain [−Mel mutants were defective in the active transport system for diphenolic compounds and phenoloxidase, which is the first enzyme needed in the L-DOPA melanogenesis pathway. A subsequent study confirmed that the loss of phenoloxidase activity was responsible for the Mel− mutant phenotype [C. neoformans virulence in vivo through disruption of the 5′ end of the lac1 gene [C. neoformans has two laccase genes, where lac2 is 75% similar to lac1, but basal transcript levels of lac2 are much lower, and mutation of the gene only induced a mild delay in melanin formation [In the early 1980s, Kwon-Chung et al. produced melanin-deficient strains  observed that pigment-less mutants have smooth-walled conidia without ornamentation, and further analysis showed that the mutant cell wall was also less electronegative and hydrophobic [C. neoformans, pore sizes were markedly reduced in pigmented cells as pore radii were estimated to be 4 and 10.6 nm for melanized and non-melanized types, respectively [C. neoformans cell wall pore size also correlated with the age of the culture, as day 4 melanin ghosts displayed the greatest porosity at 41 μL Å−1 g−1 at 16.4 Å, while day 10 melanin ghosts’ porosity was 15 μL Å−1 g−1 at 10 Å. The same study also showed that an antibody against melanin decreased the total porosity from 16 to 8 μL Å−1 g−1 [Melanin’s role in the cell wall has been extensively studied in ell wall . C. neofme older . The prerophobic . Interesectively . C. neof Å−1 g−1 , which hC. neoformans melanin is described as being composed of irregular granules 50–80 nm in diameter, and similar granular structures are observed in other fungi such as Fonsecaea pedrosoi, Hortaea werneckii, and Agaricus bisporumand [C. neoformans, these granules are cross-linked with chitin, which is a polymer of β-linked N-acetylglucosamine (GlcNAc) subunits joined in antiparallel chains by hydrogen bonding to produce strong microfibrils [In terms of its structure within the cell wall, porumand ,42,43,44ofibrils . Chitin ofibrils .chs-deficient mutants, we were able to learn how chitin interacts with melanin in Wangiella dermatitidis, C. neoformans, and Candida albicans. In W. dermatitidis, the WdCHS4 knockout mutant allowed us to observe a decrease in melanin deposition within the cell wall concomitant with an increase in the pigment’s concentration extracellularly. In C. neoformans, a similar observation occurred in cda1, cda2, and cda3 deletion mutants. These genes are responsible for the deacetylation of chitin, producing chitosan. Chitosan not only helps in cell integrity and bud separation, but the disruption of these genes resulted in a “leaky melanin” phenotype that exhibited melanin suspended in the supernatant, while still retaining some melanin within the cell [chs3 and csr2 genes, which are a chitin synthase and a regulator of chitin synthase, respectively, were also found to be necessary to retain melanin within the cell as their mutants also demonstrated the “leaky” phenotype [C. neoformans cda1∆2Δ3Δ mutants have decreased virulence, and mice inoculated with this mutant generate a strong inflammatory response followed by enrichment of Th1-type T cells in lung tissue, producing immunity against the WT strain [Through the cell . The chshenotype ,48. C. nT strain .C. albicans did not exhibit these leaky phenotypes but displayed repressed melanin externalization, and seemingly different structural types of chitin have different melanin localization effects. The chitin synthase (chs2)-deficient mutants showed accumulation of melanin grains and melanosomes within the cells, while chs8- and chs8/chs2-deficient mutants displayed WT behavior [behavior . SimilarC. neoformans produces melanin while dwelling in the soil [Melanin production enhances fungal survival in the environment. Facultative melanotic the soil . The polthe soil ,52. It ithe soil ,54. Melathe soil ,56, whicthe soil .C. neoformans to resist silver compounds is linked to its binding capacity [Cells with melanin are structurally stronger than non-pigmented cells as melanized fungi are more resistant to enzymatic degradation . The carcapacity .Melanin alters antimicrobial pharmacokinetics. In vitro efficacies of various antibiotics  and antifungals (amphotericin B and caspofungin) are significantly altered in the presence of melanin ,62,63. IC. neoformans and by 86% in an acapsular strain [C. neoformans can be affected by the surface hydrophobicity and charge [C. neoformans interferes with phagocytosis in vivo [Melanin is a negatively charged polymer . Melanizr strain . It has d charge . Microbid charge ,68. In f in vivo .C. neoformans is able to escape macrophages via nonlytic exocytosis [The mere presence of melanin, regardless of charge, improves pathogen survival. Melanized and non-melanized mutants were incubated with capsule-binding monoclonal Mab 2H1 antibodies and murine macrophages. Melanized cells conferred a 31% survival advantage over the mutant with 98.5% of the cells surviving . The autocytosis . Remarkaocytosis .Cryptococcus spp. suppress the host immune and inflammatory responses [C. neoformans melanin particles. Granulomas were seen in the liver, spleen, and lung. Granuloma formation around melanin particles resembled a foreign body reaction [C. neoformans from alveolar macrophages antifungal activities [Melanin-producing strains of esponses ,75. Rosareaction . This mireaction . Interesreaction . This sutivities .−1. The macrophage’s phagocytosis ability was attenuated significantly, along with ROS production. Regarding cytokine production, TNF-a, IL-1b, and IL-6 were suppressed [C. neoformans [Since granulomas contain a wide range of macrophage morphologies, there have been numerous investigations focused on macrophages . Indeed,ppressed . Interesoformans .C. neoformans strains 52 and 145 have opposite inflammatory reactions in experimental mouse models and these strains have been used to dissect out adaptive immune processes in cryptococcosis. The two strains differ in that strain 145 produces more melanin and strain 52 produces a stronger pulmonary inflammatory response [Not only does melanization influence macrophage activities, but melanin also modulates T cell responses. response . In a puresponse . Accordiresponse .C. neoformans [Immunization of mice with cryptococcal melanin induces a robust antibody response with significant increases in IgM and IgG levels . Moreoveoformans .C. neoformans capsule and enhance the clearance of these cells by phagocytes [The complement system is a major effector arm of host defense. Complement C3 fragments are deposited in the agocytes ,88. Inteagocytes . DepositA. fumigatus is another pathogenic fungus that undergoes melanization, and pigment formation in this species has been the subject of extensive investigation. 1,8-dihydroxynaphthalene (DHN) melanin is the major melanin found in A. fumigatus [pksP, ayg1, arp2, arp1, arb1, and arb2, ordered from upstream to downstream [A. fumigatus has many similarities to melanin from C. neoformans, as both provide protection from ultraviolet light and scavenge ROS generated by phagocytes [Albino conidia incubated with phagocytes induced a 10-fold increase in ROS production compared with WT, which suggests that the melanin increased ROS scavenging abilities in the WT conidia. Moreover, the albino mutants were more effectively killed by monocytes than wild-types [umigatus . A set ownstream . Melaninagocytes . Albino ld-types .Melanin regulates host pro-inflammatory cytokine responses by physically masking fungal pathogen-associated molecular patterns (PAMPs) from immune recognition such as that observed by the rodlets layer. Chai et al. demonstrated that albino conidia were able to generate much higher IL-6, IL-10, and TNF-α levels compared to WT. IL-6 and IL-10 had roughly 12-fold and 5-fold higher levels, respectively. The authors pinpointed that albino conidia had β-glucan and other PAMPs such as mannans more readily available to bind to dectin-1, Toll-like receptor 4 (TLR4) and Mannose receptors on peripheral blood mononuclear cells (PBMCs) . As coniAspergillus spp., A. flavus was the most effective in suppressing acidification, closely followed by A. fumigatus. Microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP) is a non-canonical autophagy pathway that is linked to certain pattern recognition receptors that trigger phagosome formation [A. fumigatus melanin inhibits calcium-calmodulin signaling on the protein Rubicon, a key regulator in the LAP pathway [A. nidulans as well [Jahn et al. observed that ΔpksP melanin-deficient conidia were more effectively phagocytosed and killed by macrophages when compared with WT conidia . Thywissormation . A. fumi pathway ,99, and  pathway . Melanin as well .A. fumigatus melanin. Bayry et al. demonstrated that DCs failed to produce cytokines TNF-α, IL-1β, IL-6, and IL-10 in response to melanized A. fumigatus conidia, and DCs treated with WT melanin ghost also failed to activate T cells [pksP, Δayg1, and Δarp2 mutants increased different amounts of acetyl-CoA, malonyl-CoA, and 1,3,6,8-THN, respectively. These mutants displayed altered cell walls with unmasked surface structures and were able to activate DCs.The effects of melanin vary on different immune cells. Dendritic cells (DCs) are not stimulated by  T cells . The groA. fumigatus and other DHN-melanized fungi, such as Cladosporium cladosporioides and Fonsecaea pedrosoi [Aspergillus. Goncalves et al. elucidated that DHN melanin blocks endoplasmic reticulum calcium/calmodulin signaling, which activates glycolysis and mammalian target of rapamycin (mTOR)-mediated defense against Aspergillusconidia [Mammals have evolved various systems to combat melanized fungal pathogens. On the surface of mouse endothelial cells, there is a melanin-sensing C-type lectin receptor (MelLec) that recognizes DHN melanin in the conidial spores of pedrosoi . The expconidia . Consequconidia . A. fumigatus can be cleared by activating the complement system [alb1/pksP that encodes polyketide synthase resulted in a significant increase in C3 binding on conidial surfaces, with an expected increase in phagocytosis by neutrophils and a decrease in virulence [A. niger melanin [t system ,107. Tsat system . Similarirulence ,109. Dir melanin .Fonseca spp. are causative agents of chromoblastomycosis, and these fungi produce large quantities of melanin. Melanized Fonsecaea monophora and cell wall-containing extracted melanin significantly decrease the expression of inducible nitric oxide synthase gene and the production of nitric oxide and enhanced non-protective Th2 responses [F. monophora enhanced the differential expression of genes related to immune responses, including the MAPK signaling pathway, demonstrating how melanization modifies pathogenesis [C. neoformans and A. niger melanin, melanized Fonsecaea pedrosoi was quickly labeled with C3, C4, and C9 complement components [esponses . Macrophogenesis . As withmponents .Histoplasma capsulatum [Paracoccidoides spp. [Coccidioides immitis [Blastomyces dermatitidis [Talaromyces marneffei [Paracoccidioides spp. through complex processes that extend beyond pigment production. Different Paracoccidioides species resist phagocytosis of yeast cells by macrophages, and this effect is associated with the degree of melanization in each strain [P. brasiliensis is also highly resistant to NO, ROS, hypochlorite, and H2O2 [P. brasiliensis and P. lutzii revealed that melanization leads to an abundance of virulence-associated proteins, including heat-shock proteins, vesicular transport proteins, adhesins, superoxide dismutases, proteases, and phospholipases, which further underscores the complex mechanisms that occur along with melanin production to subvert the host [P. brasiliensis [Several endemic dimorphic pathogenic fungi produce melanin, including psulatum , Paracocdes spp. , Coccidi immitis , Blastomatitidis , and Talarneffei . Melaninh strain ,115. Meland H2O2 . A recenthe host . As withiliensis .T. marneffei (formerly Penicillium marneffei) also utilizes melanin to avoid host defenses. Remarkably, the T. marneffei genome has 23 polyketide synthase genes and additional non-ribosomal polyketide synthase hybrid genes [2O2, and sodium dodecyl sulfate (SDS). Furthermore, melanized cells were significantly more resistant to phagocytosis and killing compared to melanin-deficient mutants [T. marneffei with tyrosine catabolism, which is essential for survival in the host cells [id genes . Mutants mutants . A secon mutants . There ist cells .Sporothrix spp., and the production of the polymer is closely linked to virulence [S. globosa leads to a reduction in antigen presentation by macrophages and facilitates the dissemination of the pathogen [Sporothrix species [S. schenckii strains may, instead, induce the formation of granuloma, which facilitates the survival of the yeast [Sporothrix complex species is protective against diverse antifungal compounds [Melanin is well described in irulence ,122,123.pathogen . Melanin species . Howeverhe yeast . Melaninompounds ,128.Mucorales spp. A recent paper from the Ibrahim laboratory identified compounds that could selectively inhibit eumelanin production by Rhizopus sp. [Melanin is purported to be a major factor in the pathogenicity of opus sp. . MoreoveThe enigmatic polymer’s structure remains elusive. Although there are synthetic melanins and certain precursors may mimic aspects of melanin’s activities, the complexities of melanin’s structure suggest that these are poor surrogates and do not represent the different forms of melanin. In fact, melanin formation in vivo is markedly different from that generated in vitro ,131. FutAlthough the formation of melanin is mysterious, its degradation is similarly unknown. The capacity for fungal melanin to persist in tissues even after the pathogen has been killed can challenge the host’s responses to nearby viable cells. Additionally, the effect of fungal melanin on other immune and non-immune cells is still elusive and should be experimentally addressed. Melanin has been suggested to have applications that could impact diverse aspects of life, ranging from remediation of nuclear contamination  to spaceA. fumigatus, C. neoformans, A. fumigatus, and dimorphic fungi such as Histoplasma capsulatum, Paracoccidioides spp., Coccidioides immitis, and Blastomyces dermatitidis. The polymer can be compared to a Swiss army knife as fungi use melanin to improve their survival in a variety of environments through an array of mechanisms. Since its discovery, our view on melanin has gradually evolved. Melanin not only helps fungi to resist radiation and enzymatic degradation in the environment, but it also helps fungi to wreak havoc in the human body. Melanin renders our antifungals less effective while preventing our innate immune system from actively clearing the fungal invaders by lowering ROS production and phagocytosis. In A. fumigatus, immune evasion also involves modulating dendritic cells. Though melanin seems like an all-powerful tool, our immune system has also created our own counter measures such as the MelLec (melanin-sensing C-type lectin receptor). Meanwhile, our macrophages are also tuned in to detect melanin. It is exciting to see what the future holds as we unravel more about the interplay between melanin and our immune system, which will likely lead to the discovery of new therapeutic targets to aid us in our ongoing struggle with pathogenic fungi.Melanin plays a major role in the pathogenesis of a wide variety of human pathogens such as


Candida glabrata is a yeast of increasing medical relevance, particularly in critically ill patients. It is the second most isolated Candida species associated with invasive candidiasis (IC) behind C. albicans. The attributed higher incidence is primarily due to an increase in the acquired immunodeficiency syndrome (AIDS) population, cancer, and diabetic patients. The elderly population and the frequent use of indwelling medical devices are also predisposing factors. This work aimed to review various virulence factors that facilitate the survival of pathogenic C. glabrata in IC. The available published research articles related to the pathogenicity of C. glabrata were retrieved and reviewed from four credible databases, mainly Google Scholar, ScienceDirect, PubMed, and Scopus. The articles highlighted many virulence factors associated with pathogenicity in C. glabrata, including adherence to susceptible host surfaces, evading host defences, replicative ageing, and producing hydrolytic enzymes . The factors facilitate infection initiation. Other virulent factors include iron regulation and genetic mutations. Accordingly, biofilm production, tolerance to high-stress environments, resistance to neutrophil killings, and development of resistance to antifungal drugs, notably to fluconazole and other azole derivatives, were reported. The review provided evident pathogenic mechanisms and antifungal resistance associated with C. glabrata in ensuring its sustenance and survival. Candida species. Each Candida species holds unique characteristics comparative to invasive potential, virulence, and antifungal susceptibility pattern [Candida species also pose a significant threat to patients in the intensive care unit (ICU) with consequential mortality outcomes. They are the most commonly associated health care reported cases [Candida infections include prolonged usage of broad-spectrum antibiotics, immunocompromised state of the host, and the use of medical devices in surgery including catheters [Candida species commonly cause invasive nosocomial infections in immunocompromised patients [albicans species suggests increasing pathogenicity of these species with varying degrees of clinical symptoms [Invasive candidiasis (IC) is a clinical condition that is not associated with a single  pattern . It is a pattern . Candidaed cases . Major ratheters ,4. Candipatients . It accopatients . The incsymptoms .Candida glabrata is an asexual, haploid yeast of the clade Nakaseomyces. It was initially named Cryptococcus glabrata. It then changed to Torulopsis glabrata in 1894, but the Candida genus was described in 1913 [Candida glabrata is a successful pathogen colonising epithelial surfaces  as healthy microbial flora with no age specificity [Candida glabrata is commonly found in the environment, particularly on flowers, leaves, surfaces, water, and soil. It is the second most frequently isolated cause of candidiasis after Candida albicans. It accounts for approximately 15–25% of invasive clinical cases [C. glabrata is the second most common species found in the United States and North-western Europe [C. glabrata among Candida species as a cause of BSI in U.S. ICUs between 1989 and 1999 in a survey showed that C. glabrata ranked second to C. albicans accounting for 20% to 24% of all Candida BSIs [C. glabrata causes substantial morbidity and mortality of approximately 40–60%, perhaps due to the inherent low susceptibility of C. glabrata to the most commonly used azoles [cificity . Candidaal cases ,12. In fn Europe . IncreasC. glabrata to reach the bloodstream is through the breach of natural barriers, such as the use of catheters, trauma, or surgery [C. glabrata is associated with old age, as reported by Zhang et al. [C. glabrata was isolated more from patients in the age group >70 years than the other age groups (58.2% vs. 41.8%) out of 193 samples collected. A switch from normal flora to the pathogenic state may occur, leading to disease setting in, ranging from superficial  to systemic with an alarming mortality rate [The usual route of  surgery . However surgery . Increasg et al. . Accordiity rate .Candida species. These include enzyme secretion, cellular adhesion, host defence evasion, and biofilm formation [Candida species-specific virulence factors such as the presence of hyphae for invasion into host tissues [Candida albicans filament exists in two distinct morphologies: hyphae and pseudohyphae. The expression of a specific gene set determines each morphology. The morphologies are critical as virulence factors occurring in most Candida species [C. glabrata appears to be independent of the morphology of the yeast as this species is incapable of hyphae formation. Despite that, C. glabrata lacks several pathogenic attributes, critical in other Candida species, including polymorphic switching [Virulence refers to the traits required for establishing a disease. However, strictly speaking, virulence factors have direct interaction and causing damage to the host cells . Changesormation . The inf tissues . Candida species ,21. Howe species  viewed twitching ,23; pathCandida albicans and C. glabrata show a significant difference in their mechanisms of virulence. Candida glabrata pathogenicity is associated with many virulence factors [C. glabrata infections is challenging due to the limited knowledge of its pathogenicity. The reduced antifungal drug susceptibility and the limited choices of effective antifungal agents are also challenging in treatment, as described by Yu et al. [C. glabrata in comparison to C. albicans, a high mortality rate associated with C. glabrata and rapidity of disease spread would argue otherwise [Candida glabrata seems to have evolved a strategy based on secrecy, evasion, and persistence without causing severe damage in murine models [C. glabrata exhibits a unique escape mechanism from the immune system and subsequently survives cellular engulfment and can resist antifungal treatment. This review summarises current information on the pathogenicity, virulence, and drug resistance mechanisms associated with C. glabrata  to destroy host tissues [CgPMU1-3) encoding phosphatase enzymes of different specificity. Accordingly, CgPMU2 was identified as analogous to the PHO5 gene found in S. cerevisiae. It serves as the phosphate-starvation inducible acid phosphatase gene. Almost all known candidal extracellular endopeptidases belong to the aspartic proteinase (Sap) class observed based on sequence analysis, proteolytic activity assay, and secretion of signal detection. Candida glabrata does not possess normal Sap genes in its genome [C. glabrata is exceptional from this rule because the cell wall is associated with serine protease, Cwp1 (ORF: CBS138)—a gelatinolytic enzyme [Secretion of hydrolytic enzymes is a significant determinant of pathogenicity in eactions . Phosphoeactions . Candida tissues . In addi tissues . The stu tissues  reporteds genome . In thisc enzyme .Candida species initiate infection through adherence to host epithelial tissue and colonisation within the host [Candida cell surface proteins involved in specific adherence to surfaces are described as adhesins, and they are critical in mediating biofilms’ formation [Candida glabrata lacks yeast-to-hyphae switching, it grows only in the yeast form, contrary to the virulent switch of C. albicans. A significant virulence factor of C. glabrata is its ability to adhere firmly to many different substrates [the host . Candidaormation . Candidabstrates .Candida species, particularly C. glabrata or C. albicans, have developed in large gene families [C. albicans are critical for the fungal adherence to host epithelial cells [C. albicans, the main adhesins useful in C. glabrata originated from the epithelial adhesin (EPA) family. These adhesins facilitate C. glabrata attachment to host epithelial cells and assist in macrophage entry [EPA family. According to the mass spectrometric analysis obtained by De Groot et al. [C. glabrata contains a unique, high number of genes encoding glycosylphosphatidylinositol (GPI) proteins from different clusters. Both (EPA and GPI) proteins are essential in adherence to human epithelial surfaces and biofilm formation. Cell wall components mediate interactions between C. glabrata and susceptible host, facilitating tissue adhesion and invasion. In addition, they are involved in biofilm formation, triggering the host immune response, and may confer resistance to antifungal drugs [C. glabrata. Thus, the cells reflected alterations of adhesion capacity and cell surface hydrophobicity.Cell surface adhesins in families . The aggal cells . Unlike ge entry . One suct et al. , 23 cellt et al.  also repal drugs ,37. NotaCandida species. In addition, the matrix contributes to pathogenicity by increasing drug tolerance and promoting immune evasion [Candida species, including C. parapsilosis, C. tropicalis, C. glabrata, and C. auris, also associate with extracellular synthesis and high rich polysaccharides contents [Biofilms are considered biological communities formed by microorganisms with a high degree of organisation, structure, coordination, and functionality encased in a self-created extracellular matrix . Accordi evasion . Biofilmcontents .C. albicans and C. glabrata can form biofilms on abiotic substrates, especially medical devices including catheters and implanted materials [C. albicans with high biofilm mass, densely packed with pseudohyphae. However, C. glabrata produces sparse biofilm (less weight) with yeast cells. Thus, it is an essential pathogenic mechanism for its survival [Both aterials ,27. Micraterials . Biofilmsurvival  . An outer layer of proteins glycosylated with mannan constitutes the pathogen-associated molecular patterns (PAMPs). The PAMPs are recognised by specific innate immune receptors known as pathogen recognition receptors (PRRs) [The fungal cell wall is the primary contact site for host-pathogen interaction . The funs (PRRs) . The cels (PRRs) . Moreoves (PRRs)  showed tC. glabrata. Deregulation of chitin biosynthesis is a potential mechanism of virulence and resistance to antifungal therapy—the presence of drugs, such as echinocandin, results in the corresponding increase in chitin synthesis. The chitin maintains the cell wall’s structural integrity, as chitin replaces β-1,3-glucan. High chitin content restricts the penetration of the drug through the cell wall [Candida glabrata presents strange features related to cell wall organisation, such as overexpression of genes encoding adhesion-like GPI-anchored proteins or the implication of GPI-anchored aspartyl proteases (yapsins) in the infection process. These features indicate key virulence factors, with multiple roles in the high tolerance to azole drugs, adhesion to susceptible host cells, or survival inside macrophages [During fungal growth, the cell wall expansion causes permanent remodelling of the polysaccharide network, consisting of mannans, β-glucans, and chitin. Chitin is a homopolymer of β-1,4-N-acetylglucosamine (GlcNAc). Chitin is essential for fungal biological functions, including cell division, septa formation, hyphal growth, and virulence . The chiell wall . Candidarophages .Candida species [Candida glabrata has well-characterised genes, including ACE2 (CgACE2), a transcription factor that serves as a negative regulator of virulence. It was studied in an invasive infection of an immunocompromised mice model. The evolved (Evo) strain is another hyper-virulent C. glabrata strain with a single nucleotide mutation in the chitin synthase gene CHS2. Both mutants have enhanced virulence. Moreover, they stimulate inflammatory response factors, such as tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Thus, the ace2 mutant and Evo strain exhibit a clumpy pseudohypha-like structure [PDR1 gain-of-function mutation, a strain with mitochondrial dysfunction, and the anp1 and mnn2 glycosylation mutants [Genetic mutations confer susceptibility to patients against  species . Candidatructure . Other s mutants .Candida glabrata requires iron as an essential micronutrient for its growth during infection. Thus, it is necessary to strategize the mechanism for its acquisition for disease establishment [3+, reductive iron procurement, and haemoglobin/haem uptake. All these frameworks are operational in C. glabrata except for the receptor-interceded haem uptake [Candida glabrata has high-affinity iron uptake mechanisms as critical virulence determinants.lishment . Among tm uptake . The und3+) and ferrous iron (Fe2+). Both ionic states have different effects on pathogenic microorganisms. For instance, Fe3+ is poorly soluble in alkaline conditions, and Fe2+ becomes toxic by promoting ROS production via the Fenton reaction [C. glabrata strains deleted for proteins  domain-containing structural proteins CgCcw14, CgMam3, and putative haemolysin) confirmed that these proteins are potentially implicated in iron metabolism.Hosts’ fundamental approach uses ‘nutritional immunity’ to limit the iron required by invading pathogenic microorganisms, similar to in humans, available iron seized by various carriers and storage proteins, including haemoglobin, transferrin, and ferritin. They virtually deprive the available iron system, leaving no option for invading organisms. It, thus, exploits other iron source mechanisms  . Iron isreaction . Accordireaction  that theSaccharomyces cerevisiae is a non-pathogenic yeast belonging to whole-genome duplication clade (WGD), having significant similarities with pathogenic C. glabrata [C. glabrata combines the iron regulation network properties of both pathogenic and non-pathogenic fungi (S. cerevisiae). Candida glabrata, such as S. cerevisiae, uses the Aft1 gene as the primary positive regulator during the sub-optimal iron condition. At the same time, Cth2 degrades mRNAs encoding iron-requiring enzymes. However, it contrasts with S. cerevisiae in that it requires Sef1 ortholog for total growth under iron-limited conditions. The iron homeostasis mechanisms in C. glabrata is still unknown. Candida glabrata showed host-specific iron acquisition mechanisms by utilising siderophores and haemoglobin as a source of iron and haemolysin. It also uses cell wall structural protein to maintain iron homoeostasis [While glabrata , it is pglabrata ,53. C. geostasis .Saccharomyces cerevisiae and is referred to as general stress response or environmental stress response (ESR). Genome-wide environmental stress response (ESR) expression profile of C. glabrata is coordinated by Msn2 which is the main transcriptional response activator. Transcription factors Msn2 and Msn4 are crucial for resistance to various stresses in C. glabrata [Yeast cells within their natural habitat make many metabolic adjustments in response to changes in extracellular environmental nutrients. Such changes result in gene expression, which are either upregulated or downregulated depending on the environmental requirements . Adaptatglabrata . Activatglabrata .Candida species can quickly adapt to host environmental changes as commensal pathogens even under nutrients bioavailability restriction [Candida species use different nutrients available in the vast host niche. The Candida pathogens possess a high degree of metabolic flexibility because of the adaptive metabolic mechanisms necessary for significant nutrient acquisition [triction ,59. Canduisition . Fungal 2 are associated with several steps during host invasion and optimal growth of Candida species [Candida species can withstand a wide range of temperatures and pH as virulence factors [Candida glabrata grows optimally at 37 °C and, therefore, thrives best in the human host and can grow at 42 °C under heat-stressed conditions [C. glabrata on the Galleria mellonella model indicated that G. mellonella only became susceptible to infection at 37 °C. Thus, this suggested that some essential genes for C. glabrata virulence are switched on only at 37 °C [Environmental parameters including temperature, pH, serum, and CO species . Candida factors . Candidanditions ,57. Tempnditions . A studyat 37 °C .C. glabrata in the presence of acetate, lactate, ethanol, or oleate reduces the growth in both the planktonic and biofilm states. The use of glucose as a carbon source, on the contrary, showed significant growth in both states. Moreover, the findings reported the necessity of isocitrate lyase (ICL1), the glyoxylate cycle gene for acetate utilisation, ethanol, and oleic acid, and partly required for the utilisation of lactate in C. glabrata.Flexibility in carbon metabolism is critical for the survival, propagation, and pathogenicity of many human fungal pathogens . AccordiC. glabrata has not been extensively investigated. The low pH of C. glabrata cultures during pyruvate production causes a slow or total halt in growth due to acid accumulation [Candida glabrata ASG1  deletion resulted in increased tolerance to salt stress [Candida glabrata makes its extracellular environment alkaline when grown on amino acids as the sole carbon source in vitro. Mutant C. glabrata that lacks fungal mannosyltransferases resulted in strictly reduced alkalinisation in vitro. The condition subjects C. glabrata to acidified phagosomal activity [C. glabrata observes low pH as less stressful than high pH [C. glabrata and C. albicans. Thus, this demonstrates the decreased efficacy of azole drugs in vitro at acidic pH [The mechanism of acid stress tolerance in mulation ,65. Contmulation  that ovet stress . Active activity . Proteom high pH . The lowcidic pH .Candida glabrata lacks hyphal formation and phagosomal extrusions to escape the phagocytic cells attack contrary to C. albicans [Cryptococcus neoformans, the produced capsules inhibit phagocytosis by macrophages and prevent the killings of the already internalised cells [Candida species. C. glabrata has high-stress resistance. Perhaps its enhanced sustenance during starvation allows it to survive and replicate inside the immune system cells (macrophages). The C. glabrata are engulfed during bloodstream circulation [ICL1 gene helps promote the growth and prolonged survival of C. glabrata during macrophage engulfment. Thus, C. glabrata shows a unique immune system evasion mechanism and survives after cellular engulfment despite the antifungal presence. Perhaps through concealment within intracellular niches [C. glabrata cells, for example, resist killing by macrophages and have developed distinct tactics for intracellular survival killing and escaping phagocytosis [During phagocytosis, the internalised microbes become lysed in lysosomes—a specialised compartment in which oxidative and non-oxidative mechanisms kill and degrade the internalised microbes . Candidaalbicans ,69. In Ced cells . The lesed cells ,69 Evideculation ,18. Chewculation  revealedr niches ,28. Lactocytosis . Followiocytosis  . In contrast, neutrophil emergence becomes typical of C. albicans [C. glabrata, it is less lethal because it provokes a low inflammatory immune response. The systemic mouse infection models indicated that even at high inocula doses of intravenous infection [C. glabrata against oxidative stress [Candida species, C. glabrata is exceptional; it does not germinate into hyphae yet is virulent [Successful clearance of pathogens depends on phagocytes’ rapid actions of the innate immune system, such as macrophages, dendritic cells, and neutrophils . The prie system . Becausealbicans . Despitenfection . Furthere stress . Considevirulent .Candida glabrata as occur in S. cerevisiae, C. albicans, and C. neoformans show a replicative ageing, a process where original mother cells progressively age, producing asymmetric mitotic divisions resulting in phenotypically distinct daughter cells [C. glabrata and a mammalian host may result in resilience and high-stress tolerance. The host becomes vulnerable to invasive diseases during neutropenic or immunocompromised states [er cells . It can er cells . A motheer cells . Severaler cells . The pheer cells . Thus, fd states .Candida glabrata can shift from a commensal to pathogenic state due to the pressure of neutrophils. Bouklas et al. [C. glabrata in the murine models. The findings indicated that ageing leads to remodelling of the cell wall and that neutrophils selection controls generational distribution within the C. glabrata population. The in vivo study by Bhattacharya et al. [C. glabrata mother cells’ large cell sizes and thicker cell walls contribute to their better resistance to neutrophil killings than the young daughter cells.s et al.  reporteda et al.  viewed tCandida species. Knowledge of C. glabrata infection symptoms is essential because Candida species commonly share indices of suspicion of the disease. C. glabrata among the non-albicans Candida species can acquire drug resistance. Moreover, it can develop secondary resistance to other available antifungal classes, resulting in poor treatment outcomes. It is a well-known fact that both C. krusei and some C. glabrata have intrinsic resistance to fluconazole. In such a situation, proper diagnosis is essential to justify appropriate treatment [The emergence of antifungal resistance becomes a problem in clinical medicine, significantly when associated with reatment .Candida species, C. albicans and C. glabrata differ significantly in response to antifungal therapy [The incidence of candidemia caused by fluconazole-resistant strains and derivatives is high . Azole d therapy . Flucona therapy ,81.Candida glabrata infections are complicated to treat due to their inherent resistance to antifungals, especially against azoles [C. glabrata has intrinsic antifungal resistance, especially to fluconazole. Arendrup and Patterson [C. glabrata developed acquired resistance to antifungal drugs through prolonged exposure. Moreover, Jensen et al. [t azoles . Sardi et azoles  viewed tatterson  argued tn et al.  supporteC. glabrata shows multi-drug-resistant capacity at an alarming rate. The genomes of C. glabrata can accumulate gene mutations that result in phenotypic resistance to antifungals after exposure to multiple drugs [MSH2 gene, encoding a DNA mismatch repair protein, occur in C. glabrata. Its effects have been found in clinical isolates to facilitate the selection of resistance to azoles, echinocandins, and polyenes in vitro [C. albicans and C. glabrata growing in biofilms settings [Moreover, le drugs . For exain vitro . On a gesettings .C. glabrata to show resistance to fluconazole in clinical isolates indicates the need to improve the diagnostic approach. In addition, it promotes new antifungal therapy for easy management of such cases [Azole drugs play a critical role in clinical practice, especially fluconazole, clotrimazole, and imidazoles . Fluconach cases .Candida glabrata possesses numerous resistance mechanisms to fluconazole, including fluctuation of gene regulation, genetic mutations, and cross-resistance among azole derivatives [Candida species, including mutations in the ERG11 gene and the proliferation of copy number of azole targets. Other mechanisms include blockage of the ergosterol biosynthesis pathway. Mutations in ERG11 and PDR1 can mediate azole resistance; daughter cells will inherit the mutations and persist [C. glabrata as observed in Iranian isolates. Resistance mechanisms are also associated with significant facilitator superfamily efflux pumps, leading to the increasing efflux of azole drugs. Although numerous possible tools have been reported previously, the exact resistance mechanism is not entirely clear on azole resistance. Approximately more than 140 alterations in the ERG11 target gene have been described. Some alterations are exclusively found in azole-resistant isolates, whereas some are obtained in susceptible isolates [ivatives . Yoo et ivatives  describe persist . Over-exisolates .Candida glabrata sensitivity to 4 Nitroquinoline N-oxide (CgSNQ2) and Candida glabrata Candida drug resistance 1 and 2 (CgCDR1 and CgCDR2) genes. More specifically, the free nitrogen atom of the azole ring binds an iron atom within the enzyme haem group. Thus, it prevents oxygen activation and causes demethylation of lanosterol that inhibits the ergosterol biosynthesis process. The inhibition is toxic methylated sterols accumulated in the fungal cellular membrane, and cell growth is arrested [The mechanism of action of azole is to target the cytochrome P450 enzyme sterol 14α-demethylase. The enzyme converts lanosterol to ergosterol as an essential structural component of the fungal cell membrane . Accordiarrested .C. glabrata at high frequency. Several genes on chromosomes (ChrEL and ChrFL) potentially mediate interactions between C. glabrata and the susceptible host. Duplicated segments of ChrFL encode a transporter of the ATP-binding cassette family (CAGL0F01419g) that is very similar to S. cerevisiae AUS1. The small chromosome F encodes an ortholog of S. cerevisiae ABC transporter PDR5 (CAGL0F02717g) known in C. glabrata as PDH1. Torres et al. [AUS1 and PDH1 is also observed in C. glabrata-resistant isolates [According to Poláková et al. , the gens et al.  viewed tisolates . DuplicaC. glabrata cells [The major facilitator superfamily (MFS) is a membrane transporter that helps in accomplishing the active efflux of azole. MFS transporters facilitate enhanced fluconazole efflux particularly in ageing ta cells .C. glabrata for azole resistance through the upregulation of ATP-binding cassette (ABC) transporter genes. The upregulation of these transporters is associated with gain-of-function (GOF) mutations in the transcriptional regulator encoded by CgPDR1. Cells with mitochondrial DNA deficiency are called ‘petite mutants’ [C. glabrata isolates (BPY40 and BPY41) obtained from the same patient on different occasions. The former was azole sensitive, while the latter was azole-resistant. Upon testing, BPY41 showed mitochondrial dysfunction compared to BPY40. The virulent analyses, based on mortality and fungal tissue burden in both systemic and vaginal murine infection models, suggested higher virulence of BPY41 than BPY40. Then, oxido-reductive metabolism and the stress response were also observed in the BPY41 isolate. Based on the microarray analyses, some genes responsible for cell wall remodelling were upregulated in BPY41 compared to BPY40. These pieces of evidence suggested that virulence and resistance to azole were linked to mitochondrial dysfunction in BPY41.Mitochondrial DNA deficiency is another mechanism used by mutants’ . Ferrarimutants’  reportedC. glabrata isolates with MICs of ≥8 µg ml−1. One C. glabrata isolate showed MICs of ≥1 µg mL−1 and two C. glabrata isolates showed to have MICs of ≥1 µg mL−1. Song et al. [C. glabrata isolates tested were resistant to fluconazole. The five isolates were resistant to itraconazole. Similarly, all the isolates were resistant to itraconazole. In a similar resistance pattern, Premamalini et al.’s [C. glabrata isolates were resistant to fluconazole and itraconazole. However, the isolates were susceptible to voriconazole. A study conducted in China reported that 12.2% of C. glabrata are resistant to fluconazole. The result also showed a 17.8% susceptibility of the standard strains to voriconazole [Candida species using broth microdilution method is ≤8 µg mL−1 for susceptible (S), 16–32 µg ml−1 for susceptible-dose dependent (S-DD), and ≥64 µg mL−1 for resistant (R) [For instance, Nedret Koc et al.  reportedg et al.  reportedet al.’s  study coconazole . These fconazole  that newtant (R) .C. albicans and C. glabrata in suspected severe IC conditions [C. glabrata with reduced susceptibility to fluconazole Colombo et al. [C. glabrata usually correlates with high azole and frequent echinocandin usage in hospitals or specific hospital wards [Candida species strains resistant to first-line antifungals (such as fluconazole and echinocandins) are increasingly documented. Echinocandins exist according to the international guidelines in three available types . They differ based on the route of metabolism, half-life, and safety [Echinocandins are recommended as first-line therapy for non-neutropenic patients associated with nditions . Echinoco et al. . The emeal wards . Candidad safety .d-glucan synthase, an enzyme necessary for cell wall synthesis. They have excellent fungicidal activity against most Candida species [Candida strains remains low, with around 4% observed in C. glabrata and less than 1% in C. albicans [Candida parapsilosis [Candida species, particularly C. glabrata [C. glabrata or C. krusei, echinocandins offered preference over azoles. One major setback of echinocandins oral administration is the inadequate bioavailability, and therefore, it is often given intravenously [Candida species susceptibility [C. glabrata. The resistance acquired through mutation (amino acid changes) of the critical regions in FKS1 and FKS2 genes encode β-1,3-d-glucan synthase, the potential target enzyme echinocandins [The echinocandins drugs act by inhibiting β- species . The prealbicans . Intrinspsilosis . Howevervenously . The fretibility . Despiteocandins ,103.FKS2 relative to FKS1 [C. glabrata and less than 1% for C. albicans. However, Sasso et al. [C. glabrata-resistant strains, significantly associated with FKS1 and FKS2 gene mutations. Mutations in two hotspot regions (HS1 and HS2) of these genes have been recognised as the primary mechanism for echinocandin resistance [Candida isolates showed resistance to caspofungin. All isolates were highly susceptible to anidulafungin except C. glabrata with 10% resistance. The SENTRY surveillance program between 2006 and 2010 reported 11% echinocandin and fluconazole resistance among C. glabrata  [Mutations frequently occur in  to FKS1 . Shields to FKS1  reportedo et al.  reportedsistance  findingse., MDR) .Candida species. It is used in the pharmacotherapy of life-threatening fungal infections [Candida species. However, they differ based on the daily dosing regimens and toxicity profiles. Amphotericin formulations are the best therapeutic option, mainly in catheter-related bloodstream infections in neutropenic patients [Amphotericin B (AmB) is a fungicidal polyene and has shown promising activity against many fections . Despitefections . Most ofpatients .Candida species [ERG2, ERG3, ERG5, ERG6, and ERG11 genes with the depletion of ergosterol as a significant cause of AmB resistance [C. glabrata isolates demonstrated similar MIC50 (0.25 μg/mL) against AmB for biofilm and planktonic cells. The findings attributed lower resistance of C. glabrata with biofilms against amphotericin and not about the low biofilm content of the isolates tested. The findings agreed with the study reported by Al-Dhaheri and Douglas [C. albicans, C. krusei, and C. parapsilosis after exposure to amphotericin. Such a ‘persister’ population was absent from the biofilms of C. glabrata. In contrast, Rodrigues et al. [C. glabrata can produce biofilms in the presence of AmB therapeutic concentrations due to the high concentrations of carbohydrate and β-1,3 glucan on the biofilm matrices. This underlines the capacity of Candida cells to rapidly adjust to external aggressions. Thus, this suggests why patients undergoing AmB therapy may still manifest resilient Candida infections. According to the findings of Bhattacharya et al. [C. glabrata causes higher tolerance to killings by AmB and micafungin due to the higher transcription of glucan synthase gene, FKS1. The study of Aslani et al. [90 values of 4 μg/mL. According to the findings reported by Norimatsu et al. [C. glabrata and C. parapsilosis bloodstream infections in the case of an 80-year-old woman.The mechanism of action of AmB is to bind to ergosterol in the plasma membrane resulting in the leakage of cytoplasmic materials and cellular destruction ,98. The  species . Some stsistance . Tay et sistance  reported Douglas  that ‘pes et al.  viewed ta et al. , replicai et al.  reportedCandida species to become potentially life-threatening human pathogens. Management of candidiasis involves the identification and control of host predisposing factors to infection. Moreover, Candida species possess many virulence factors, including secretion of hydrolytic enzymes host cell wall adherence, and biofilm formation. The factors help to increase their persistence and survival. Candida glabrata further adopt strategies of evading the action of immune cells. Candida glabrata cells become engulfed in the macrophages. They quickly adopt other mechanisms of survival by undergoing massive division and release upon rupture. Thus, it is essential to study the functions of those genes associated with that mechanism because of increasing mutation occurrence. Constant surveillance of antifungal susceptibilities in clinical isolates of C. glabrata at the national and international levels is necessary to control the spread of resistance. The survey will provide practical strategies for the prophylaxis and treatment of human infections associated with C. glabrata. Moreover, acquired and intrinsic resistance to fluconazole and rapid resistance development to a few available antifungal drugs are of significant clinical concern. These issues prompt the urgent development of a better diagnostic approach to detect and identify C. glabrata for effective and timely treatment and management. Diagnostic development of methods with robust sensitivity, specificity, and short turn-around time could significantly assist to better manage patients with candidiasis caused by C. glabrata.The alteration of host interaction factors facilitates opportunistic harmless commensal


Echinococcus multilocularis. MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro, determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E. multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E. multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E. multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE.The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite  Echinococcus multilocularis. Current treatment requires surgery and/or prolonged drug therapy. Thus, novel strategies for the treatment of AE are needed. MicroRNAs (miRNAs), a class of small ~22-nucleotide (nt) non-coding RNAs with a major role in regulating gene expression, have been suggested as potential therapeutic targets for treatment and control of helminth parasite infections. In this work, we analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro. We predicted functional roles for highly expressed miRNAs and found that they could be involved in essential roles for survival and development in the host. We determined that E. multilocularis miR-71, a highly expressed miRNA that is absent in the human host, is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. Germinative cells are a relevant cell type to target for anti-echinococcosis drug development. MiRNAs that are absent in the human host, involved in essential functions, highly expressed and/or expressed in germinative cells in E. multilocularis metacestodes may represent selective therapeutic targets for treatment and control of AE.Alveolar echinococcosis (AE) is a zoonotic disease caused by the metacestode stage of the helminth parasite  Echinococcus multilocularis and Echinococcus granulosus are the etiological agents of alveolar echinococcosis (AE) and cystic echinococcosis (CE), respectively. These diseases are among the most severe parasitoses in humans and belong to a group of priority neglected zoonotic diseases identified by the World Health Organization (WHO) (https://www.who.int/neglected_diseases/zoonoses/infections_more/en/).The small tapeworms E. multilocularis (fox tapeworm) is complex and involves several developmental transitions. Adult worms develop in the small intestine of definitive hosts (mainly foxes) and produce infective eggs containing the oncospheres, which are released with the host faeces into the environment. The infective eggs ingested by the intermediate hosts  release the oncosphere that after penetrating the intestinal epithelium develops into the metacestode larval stage in host internal organs, with almost all primary infections occurring in the liver . Re. Re84]. E. multilocularis in vivo metacestodes [E. multilocularis primary cells PC1 [E. canadensis metacestodes [Drosophila spp. [S. mansoni [Another highly expressed miRNA in metacestodes samples, MCvivo, MCvitro and MCana, was miR-4989. This miRNA was also highly expressed in primary cells samples, PC1. MiR-4989 (miR-277 family) was found to be highly expressed in cestodes , E. multells PC1  and E. ccestodes . MiR-498ila spp. . In addi mansoni .E. canadensis and E. granulosus s. s. [E. multilocularis [E. granulosus s. s. [One of the most expressed miRNAs in protoscoleces samples, naPS and PS, was miR-1. This miRNA was found to be highly expressed in protoescoleces samples from us s. s. . MiR-1 ius s. s. . The higocularis ,87 and ius s. s. .mir-71/2b/2c and mir-277a/4989, we found different levels of miRNA expression between miR-71 and miR-2b/miR-2c and between miR-4989 and miR-277a , with the only exception of PC3_2 . For difAlthough the biological replicate aPS_1 showed a high level of correlation, this replicate was excluded for differential expression analysis due to incorrect clustering Figs  and 3E. multilocularis miRNA expression throughout metacestode development in vitro we performed differential expression analyses of all identified miRNAs between different sample types representing the following in vivo transitions or in vitro cultured conditions: i) metacestode development from primary cells (PC1 to PC2-3), ii) mature metacestode development from primary cells (PC2-3 to MCana), iii) primary cells isolated from mature metacestodes (PC1 vs MCana), iv) protoscolex development from metacestodes (MCvitro to naPS), v) host-parasite interaction (MCana vs MCvivo), vi) metacestode metabolism (MCvitro to MCana) and vii) activation of protoscoleces (naPS to aPS).To analyze We found significant differences on miRNA expression for the following comparisons: PC1 to PC2-3, PC2-3 to MCana, MCvitro to naPS and MCana vs MCvivo .E. multilocularis [E. multilocularis metacestode cyst wall [We found 15 miRNAs differentially expressed between PC1 and PC2-3 , suggestocularis . Anotherocularis ,84. Fiveocularis . Higher yst wall ,91,92.Bombyx mori [We found ten miRNAs differentially expressed between PC2-3 and MCana , suggestbyx mori . Anotherbyx mori . Seven mE. canadensis [E. multilocularis, it shows further complexity and similarity to that of the adult worm in the protoscolex stage [We found nine miRNAs differentially expressed between MCvitro and naPS , suggestnadensis . Interesnadensis . Althougex stage .in silico functional analysis section). Among them were a tetraspanin [in vitro from the trematode S. mansoni. Also, miR-71 was detected in sera of S. mansoni infected mice [in vitro from Taenia crassiceps [E. multilocularis metacestodes [S. japonicum [E. multilocularis metacestode lives long term in their host, it would be interesting to determine whether bantam is also involved in modulating the host immune response. However, more experiments are needed in order to determine whether this and the other differentially expressed miRNAs might play a role in host-parasite interaction.We found seven miRNAs differentially expressed between MCana and MCvivo . Since traspanin  and Merlraspanin . Interested mice . In addited mice . Also, massiceps  and verycestodes , suggestaponicum . Considein vivo transitions or in vitro cultured conditions suggests that post-transcriptional regulation of particular sets of mRNA targets may play an important role in the E. multilocularis metacestode development and maintenance of specific features in each developmental stage or in vitro cultured condition. This result agrees with the differential expression of protein coding genes found between different sample types in E. multilocularis [in vitro iii) may not participate during protoscoleces activation. We hypothesize that other gene regulation processes, such as transcription regulation by transcription factors, may drive these in vivo stage transitions or in vitro cultured conditions that were compared.The high proportion of differentially expressed miRNAs found between sample types representing different ocularis ,36. Howein situ hybridization aids in elucidating miRNA roles in molecular and biological processes [E. multilocularis protoscoleces [Schistosoma adult worms [in situ hybridization (WMISH) to determine the cell-specific expression of miR-71 in E. multilocularis metacestodes cultured in vitro (MCvitro)  . This miMCvitro) . We hypoE. multilocularis in vitro metacestodes  of cestodes .3. No flcestodes . Regardicestodes .2. This E. multilocularis cell types present in metacestode vesicles was incubated in vitro for a short period of time (5 hours) with the thymidine analog EdU. The germinative cells were the only cells that incorporated the proliferation marker, whereas no other morphologically differentiated cells, such as tegumental cells, glycogen/lipid storage cells and calcareous corpuscle cells did. This result indicated that EdU incorporation is a mark of germinative cells and that these cells are the only cells able to proliferate. Here, in order to determine whether miR-71 is expressed in germinative cells, E. multilocularis in vitro metacestodes were incubated with EdU before performing WISH. We found that several cells that showed EdU incorporation also showed miR-71 expression, confirming the hypothesis that miR-71 is expressed in germinative cells of E. multilocularis in vitro metacestodes . Analyses of predicted functions of miR-71 targets performed in our previous work [In addition, we found cells that showed miR-71 expression but without EdU signal .3. Althoous work  suggest ous work –28.E. multilocularis samples we performed a miRNA target prediction using miRanda algorithm . Although this number represents only more than a half of the total number of 3’UTRs , they are a confident set of 3’UTRs annotated based on the integration of RNAseq data and genomic data. A similar approach for miRNA target prediction using only a subset of 3’UTRs of the total number of coding genes was recently performed in E. canadensis [S. mansoni [E. multilocularis unique target genes, since six target genes: EmuJ_000109300, EmuJ_000484900, EmuJ_000778300, EmuJ_000808600, EmuJ_000189200 and EmuJ_000935200 had two sites for the same or different miRNAs. We found that each miRNA had a different number of predicted target genes. The number of unique targets per miRNA ranged from 11 to 165 depending on the miRNA. MiR-71 was the miRNA with the highest number of targets ~55% (165/303), whereas miR-4989 was the miRNA with the lowest number of targets ~3.6% (11/303)  . The higaponicum .E. multilocularis 3’UTRs used as input for miRNA target prediction  and the set of 6,227 ediction . ConsideE. multilocularis by both algorithms across the closely related species E. granulosus and T. solium. We found that ~75% (206/275) of the miRNA target sites were conserved in at least one species adding weight to the miRNA target predictions obtained (E. granulosus ~ 70% (193/275) than for T. solium ~ 32% (89/275) (We determined the evolutionary conservation of the 275 miRNA target sites predicted in obtained . As expe(89/275) .E. multilocularis coding genes have no annotation and/or associated GO terms [In order to strengthen the miRNA target prediction functional information, we performed a GO functional enrichment analysis of predicted miRNA targets for each individual miRNA. We did not find significant enriched GO terms for any miRNA . We hypoGO terms  and the We also carried out a KEGG pathway analysis and assigned the predicted miRNA target genes to KEGG pathways .E. granulosus or T. solium) iii) assigned gene function iv) potentially related to a known function for the same miRNA in other organisms  prediction with both algorithms ii) evolutionary conservation in at least one species  that is involved in the regulation of actin cytoskeleton as a putative target of emu-miR-1 conserved in T. solium. This result agrees with the high expression of emu-miR-1 in E. multilocularis protoscoleces. In addition, we found an Activin gene from the TGF-beta signaling pathway, which was conserved in E. granulosus. Interestingly, the Activin gene is involved in immunomodulation in E. multilocularis [MiR-1 is a highly conserved miRNA known for its role in muscle development. Among the selected targets for emu-miR-1 we found genes encoding cytoskeleton proteins such as light and heavy dyneins which were conserved in ocularis .C. elegans cah-1 that is expressed in different neurons and head ganglion. Another target of emu-miR-9 was the gene Peregrin, a putative histone modification protein. Both targets were conserved in E. granulosus.MiR-9 is a deeply conserved miRNA across evolution known to be involved in neural development. Among the selected targets for emu-miR-9 we found a gene encoding a carbonic anhydrase, which is the ortholog of Drosophila. Recently, miR-4989 was shown to be involved in development of juvenile worms in S. mansoni [E. granulosus.MiR-4989 is a divergent member of the miR-277 family. This protostomian-specific family is known to be involved in amino acid catabolism in  mansoni . Among tEchinococcus, known to be essential for temporal development in C. elegans. Among the selected targets for emu-let-7 we found a nuclear receptor that may be potentially involved in transcription regulation and a homeobox protein, which is the ortholog of C.elegans lin-39, a homeodomain protein involved in cell fate specification, regulation of transcription and development. Also, we found that transcription factor sum, the ortholog of C. elegans hlh-1, a transcription factor required during embryogenesis for proper bodywall muscle development and function, had a miRNA target site for emu-let-7. The three selected targets were conserved in E. granulosus and T. solium.Let-7 is a bilaterian miRNA, highly divergent in C. elegans. Among selected targets of emu-miR-71, we found genes that may be involved in cell cycle in E. multilocularis such as an Aurora kinase A, a mitotic checkpoint serine:threonine protein and cyclins D1 and G2 that mapped to different pathways related to cell growth and death. Interestingly, we found a potential target of emu-miR-71, Polycomb group RING finger protein 2 that mapped to signaling pathways regulating pluripotency of stem cells, conserved in both closely related species E. granulosus and T. solium. These results are in coincidence with the expression of emu-miR-71 in germinative cells found in this work. Other relevant targets of miR-71 that may be involved in regulation of lifespan and aging were a Regulatory associated protein of mTOR, Target of rapamycin complex 2 subunit MAPKAP1 and a Mitochondrial import inner membrane translocase subunit that mapped to mTOR signaling pathway or longevity regulating pathway. Evolutionary conservation of E. multilocularis miR-71 targets in E. granulosus and T. solium is shown in MiR-71 is bilaterian miRNA absent in vertebrates that is known to be involved in promotion of longevity and neuronal asymmetry in E. multilocularis PC1 cells with inverse expression between the miRNA and the target after knocking down miR-71 [E. multilocularis PC1 cells from our previous study [-miR-71 targets predicted here , naPS: non-activated protoscoleces, aPS: activated protoscoleces.MCvivo: metacestodes extracted from experimentally infected jirds, MCvitro: metacestodes cultured (TIF)Click here for additional data file.S2 FigE. multilocularis samples based on normalized miRNA read counts. Each dot indicates one biological replicate from a given sample. The dot corresponding to the biological replicate aPS_1 is indicated. MCvivo: metacestodes extracted from experimentally infected jirds, MCvitro: metacestodes cultured in vitro in aerobic conditions, MCana: metacestodes cultured in vitro in axenic and anaerobic conditions, naPS: non-activated protoscoleces, aPS: activated protoscoleces, PC1: primary cell cultured in vitro for 48 hs, PC2: primary cells cultured in vitro for 7 days and PC3: primary cells cultured in vitro for 21 days.PCA plot of all biological replicates from different (TIF)Click here for additional data file.S3 FigPearson’s correlation coefficients are shown for each pair of biological replicates in the upper panels. MCvivo (A). MCvitro (B). MCana (C). PC1 (D). PC2 (E). PC3 (F). naPS (G). aPS (H).(TIF)Click here for additional data file.S4 FigPearson’s correlation coefficients are shown for each pair of biological replicates in the upper panels.(TIF)Click here for additional data file.S5 FigGerminal layer with a brood capsule showing nuclear labeling with DAPI  and scrambled LNA probe as negative control  (A). Germinal layer with a brood capsule showing nuclear labeling with DAPI  and miR-71 expression  (B).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S1 FileGerminal layer showing proliferating cells with EdU  with and without expression of miR-71 .(AVI)Click here for additional data file.S2 FileAll mapped reads were BLAST searched  against this in-house database for classification into categories.(FA)Click here for additional data file.


Aedes mosquitoes. Four main classes of insecticides are widely used for mosquito control: organochlorines, organophosphates, pyrethroids and carbamates. Here, we review the distribution of dengue fever from 2000 to 2020 and its associated mortality in Southeast Asian countries, and we gather evidence on the trend of insecticide resistance and its distribution in these countries since 2000, summarising the mechanisms involved. The prevalence of resistance to these insecticides is increasing in Southeast Asia, and the mechanisms of resistance are reported to be associated with target site mutations, metabolic detoxification, reduced penetration of insecticides via the mosquito cuticle and behavioural changes of mosquitoes. Continuous monitoring of the status of resistance and searching for alternative control measures will be critical for minimising any unpredicted outbreaks and improving public health. This review also provides improved insights into the specific use of insecticides for effective control of mosquitoes in these dengue endemic countries.Dengue fever is the most important mosquito-borne viral disease in Southeast Asia. Insecticides remain the most effective vector control approach for The online version contains supplementary material available at 10.1186/s13071-021-04785-4. Aedes mosquitoes  are the main vectors for several diseases associated with arboviruses, such as dengue, dengue haemorrhagic fever, dengue shock syndrome, yellow fever, chikungunya and Zika virus infection. There are two medically important species of Aedes mosquitoes that are associated with the transmission of dengue virus: Aedes aegypti  and Aedes albopictus . The adults of Ae. aegypti and Ae. albopictus are both black in colour but they can easily be differentiated by the pattern of white scales on their dorsal side of the thorax: Ae aegypti has two straight lines surrounded by curved lyre-shaped lines on the side while Ae. albopictus has a single broad line of white scales at the middle of the thorax  = 1) but showed moderate resistance to temephos and spinosad (RR < 5). These results suggest that Bti, diflubenzuron and pyriproxyfen may be used as alternative larvicides for dengue vector control in water-storage containers in Laos at places with temephos-resistant mosquito populations.Marcombe et al.  conducte. [Ae. aegypti mosquitoes were resistant to permethrin. In particular, Ae. aeygpti from Bandar Baru Bangi (S15) exhibited higher knockdown rate of 600-folds compared with laboratory strains [. [Ae. aegypti. All three field-collected strains exhibited strong resistance to pyrethroids with complete absence of mortality but were highly susceptible to OPs. Similarly, Ae. aegypti larvae collected in Selangor and Penang also showed same resistant patterns [Ae. aegypti populations were also resistant to DDT and carbamate bendiocarb. Resistance profiles were associated with kdr mutations in Malaysian Ae. aeygpti populations. Most of these strains harboured the F1534C, V1016G and V1023G substitution alone or combination mutations of V1023G and S996P [As of 12 December 2020, a cumulative 88,074 dengue cases had been reported in Malaysia, which marks a drastic decrease compared with the 124,777 cases for the same period in 2019 . Studies.  conducte strains . Rosilawrains [.  extendedpatterns –155. Othnd S996P .Ae. albopictus populations, secondary vectors of dengue, were found to be mostly fully susceptible to pyrethroids, with Kuala Lumpur strains showing a moderate tolerance to deltamethrin and permethrin [Ae. albopictus to DDT, bendiocarb, dieldrin and malathion in several states was reported and both Aedes species exhibited elevated levels of CYP and oxidase enzymes [CYP6P4 in Ae. albopictus and CYP6P12 in Ae. aegypti as being associated with pyrethroid resistance whereas CYP6N3 was observed across DDT- and carbamate-resistant Ae. albopictus populations. Several CYP genes, including CYP9J27, CYP9J26, CYP9J28, CYP9M6 and CYP6CB1, were found to be overexpressed in pyrethroid-resistant Ae. aegypti [Ae. albopictus populations [On the other hand, rmethrin . In that enzymes . Ishak e enzymes  reported aegypti . Overexpulations .Ae. albopictus was reported in Sabah, particularly on the West Coast and in Kudat were strong resistance to DDT and malathion with complete survival, and to tempehos and bromophos with mortality rates ranging from 0 to 93.33% were observed [Ae. albopictus populations were susceptible to pyrethroids but displayed moderate resistance against the other three classes of insecticides in Sabah [Ae. albopictus populations in Sabah could be explained by its geographical landscape of tropical rainforests, which is a favourable habitat for Ae. albopictus.Research on insecticide resistance in East Malaysia is limited. Larvicide resistance in observed . Adult Ain Sabah . The preAe. aegypti were reported in Myanmar. F1534C mutations were detected in permethrin-resistant Ae. aegypti in Yangon City [kdr mutations were observed in pyrethroid-resistant Ae. aegypti [Aedes mosquitoes collected at seven townships in Yangon City where 1.2 metric tons of DDT had been employed for dengue fever control until it was banned in 2003 [The Ministry of Health and Sports reported 4121 cases of dengue fever with 32 deaths across Myanmar as of 11 July 2020 . A smallgon City . Single  aegypti . Three p aegypti . A genot aegypti . DDT res in 2003 .Aedes aegypti collected from Mandaluyong City were only susceptible to malathion but resistant to all other insecticides [Aedes mosquitoes in Philippines.In the Philippines, 420,000 dengue cases were reported in 2019, of which 1565 were fatal, which is double the number of cases reported the previous year for the same period [cticides . To dateAedes mosquitoes as of 2 February 2021, which twofold lower than the number of cases during same period of the previous year [Ae. aegypti mosquitoes in Singapore were reported to be resistant to permethrin and cypermethrin decades ago [Ae. aegypti larvae and adult mosquitoes has been reported in Singapore [Vssc mutations were detected in pyrethroid- and DDT-resistant Ae. aegypti carrying the V1016G, F1534C and F1269C substitutions [CYP genes, i.e. overexpression of CYP6BB2, CYP9M6, CYP9M4, CYP9M5, CYP4C50, CYP6Z7, CYP6Z8 and CYP6F3, and permethrin resistance was detected [Ae. albopictus [Ae. albopictus populations [The National Environment Agency of Singapore disclosed a total of 701 dengue cases transmitted by ous year . Ae. aegades ago . Similaringapore , 165. Vsitutions , 128, wiitutions . A corredetected , 165. F1bopictus . An assoulations .Ae. aegypti populations in Thailand reported to be resistant to a wide range of insecticides, including deltamethrin, permethrin, fenitrothion, temephos, propoxur, DDT, cyfluthrin and alpha-cypermethrin [Vssc mutation is the major mechanism of pyrethroid resistance, in which V1016G was detected to be associated with the S989P and F1534C substitution in the homozygous form [CYP9J32, CYP6Z8, CYP9M9, CYP6AH1, CYP4H28), GSTs (GSTE2) and carboxylesterases  detected in resistant Ae. aegypti [Ae. albopictus samples collected from Pong Nom Ron showed high resistance to all five pyrethroids, with mortality rates ranging from 34.4 to 68.6%. Rayong strains also showed resistant to permethrin with a 51% mortality rate [Thailand reported 129,906 dengue cases in 2019 . The susrmethrin , 168–170ous form , 166. Adous form , 171. Anous form . Metabol aegypti , 174. Aeity rate .Ae. aegypti populations from Dili were found to be resistant to permethrin, lambda cyhalothrin and resmethrin in association with the overexpression of esterases [Timor-Leste reported 837 dengue cases in 2017, of which two were fatal . Insectisterases .vs 314,468 cases and 54 deaths in 2019) [Ae. aegypti to DDT and pyrethroids was first reported in 1999 [kdr mutations and overexpression of CYP enzymes in resistant Ae. aegypti populations were observed. Ae. aegypti resistant to permethrin has been reported in several provinces, including Nha Trang, Hanoi, Ho Chi Minh, Kien Giang, Dong Nai and Dak Lak, with mortality rates ranging from 3.03 to 52.25% [Ae. aegypti mosquitoes were found to be resistant to lambda-cypermethrin, cyfluthrin, etofenprox, DDT and alpha-cypermethrin [Ae. albopictus populations from Hanoi [Dengue infections have increased substantially in Vietnam with 121,398 reported cases and 19 deaths as of 29 November 2020 (in 2019) . The res in 1999 . kdr muto 52.25% . In addirmethrin , 179. Sermethrin , 180. V1om Hanoi .To date, there is no or limited access to dengue data and resistance for Brunei.Mesocyclops longisetus and M. thermocyclopoides which prey on the young mosquito instars, could be an alternative control strategy[Ae. aegypti but it is challenging to apply the copepods as most of the larval habitats are not favourable habitats for these copepods [Although insecticides were once effective in controlling mosquito-borne diseases, the increasing trends of mosquito-borne diseases may indicate an increasing resistance to or ineffectiveness of insecticides in controlling the transmission of the diseases. Furthermore, insecticides may also significantly influence the environment and ecosystems. It may be wise to revisit the concept of using chemical insecticides for controlling or eliminating mosquitoes and hence disease transmission. Long-lasting insecticide nets (LLINs) and indoor residual spraying, the use of which has been implemented as public health intervention tools for mosquito control, now require more diversified products due to the overwhelming development of insecticide resistance among mosquito populations. Biological control strategies which target different stages of the mosquito life-cycle, such as the use of numerous copepods, including  strategy, 182. Incopepods , 184. Otcopepods . Bti which destroys the gut of the mosquito larvae by producing δ-endotoxin [Metarhizium anisopliae, to adult mosquitoes [As biocontrol agents, entomopathogenic fungi, bacteria and viruses have been developed to specifically kill mosquitoes. The most commonly used microorganism isndotoxin . Severalsquitoes . The funsquitoes .Wolbachia sp., an endosymbiotic bacteria [Wolbachia sp. causes sterility, thereby suppressing mosquito populations. The presence of Wolbachia strain, wMelPop, reduces the adult lifespan via the inhibition of pathogen replication as well as the upregulation of immune genes [ via genetically engineered sterile male mosquitoes, such as OX513A . These released sterile males subsequently mate with wild females, and the resultant offspring will die before adult metamorphosis which reduces their reproductive potential. Ultimately, field trials and further research on the sustainability and cost-effectiveness of both approaches will be necessary.Another method includes the release of genetically modified mosquitoes that have been infected with bacteria  and of sbacteria . The cytne genes , 192. ThAedes mosquitoes in these endemic regions.In summary, the prevalence of dengue fever and increasing trend of resistance towards different categories of insecticides are alarming in many Southeast Asian countries. A well-researched understanding of the mechanism of resistance and susceptibility of the mosquitoes is of utmost importance for the development of an effective control method of Additional file 1: Table S1. Summary of characteristics data of the included studies.


Saussurea medusa is an important traditional Tibetan medicinal plant in China. In this study, we assembled the complete chloroplast (cp) genome of S. medusa. The complete S. medusa chloroplast genome is a circular molecular structure of 152,257 bp in length with coding GC 37.93%, consisting of two inverted repeats  separated by a large single-copy region  and a small single-copy region . The complete chloroplast genome of S. medusa contained 130 genes, including 87 protein-coding genes, 35 tRNA genes, and eight rRNA genes. Phylogenetic analysis shows that S. medusa is most closely related to Saussurea inversa and Saussurea pseudoleucoma. The complete chloroplast genome sequence of S. medusa facilitates the phylogenetic studies of Asteraceae. Saussurea medusa Maxim.  of Saussurea in China  Autonomous Region, China . The voucher samples (542122190722379LY) were deposited at the Herbarium of the Chongqing Academy of Chinese Materia Medica , Chongqing, China. Genomic DNA was extracted by using the modified CTAB method  separated by a large single-copy region  and a small single-copy region . The complete chloroplast genome of S. medusa contained 130 genes, including 87 protein-coding genes, 35 tRNA genes, and eight rRNA genes.The complete S. medusa, the phylogenetic tree was generated by the maximum likelihood (ML) method from alignments created using the MAFFT v7 . As shown in the phylogenetic tree (S. medusa is most closely related to S. inversa and S. pseudoleucoma. The result was consistent with the traditional plant morphological taxonomy. The complete chloroplast genome sequence of S. medusa facilitates the phylogenetic studies of Asteraceae.To investigate the phylogenetic relationship of tic tree , S. medu


Lithocarpus hancei (Benth.) Rehd is a widely distributed evergreen tree with broad-leaves that dominates the lower stories of the forest in China. Here, we sequenced and assembled the complete chloroplast genome of L. hancei. The genome is 161,304 bp with one large single copy , one small single copy , and two inverted repeat (IR) regions . It contains 117 genes, including 80 protein-coding genes, 33 tRNA genes, and four rRNA genes. Phylogenetic analysis of 21 representative cp genomes of the Fagaceae suggests Lithocarpus is monophyletic with strong bootstrap support and also that L. hancei is closely related to L. polystachyus. The cp genome is important for constructing a robust phylogeny of Lithocarpus and Fagaceae for future study. Lithocarpus Blume is a large genus classified in the Fagaceae with more than 300 species that is predominantly distributed in Asia (Huang and Bruce Bartholo 1999). China hosts 122 Lithocarpus species (Huang and Bruce Bartholo Lithocarpus hancei (Benth.) Rehd 1919 is a widely distributed evergreen tree with broad-leaves that occurs under 2600 m in central and south China  A.Camus has been fully annotated. To better understand the phylogenetic position of L. hancei, in this study we sequenced and assembled its chloroplast genome.s L. Cao . Chlorops L. Cao , becauses L. Cao . Howeverzengfeiy@scbg.ac.cn). Genomic DNA was extracted from silica gel-dried leaves using the CTAB method  were aligned using MAFFT with auto alignment strategy  was collected from Baishanzu Mountain, Zhejiang, China  and deposited at the herbarium of South China Botanical Garden (SCBG, Feiyan Zeng, od Doyle , and seqod Doyle . The annod Doyle  and manuL. hancei is 161,304 bp in length with a GC content of 36.7%. The quadripartite structure of the cp genome has one LSC region of 90,585 bp, one SSC region of 18,959 bp, and a pair identical IR regions of 25,880 bp. A total of 117 genes were annotated including 80 protein-coding, 33 tRNA and four rRNA genes. Comparison of the L. hancei cp genome with the publicly available L. balansae genome using the program mVISTA (Mayor et al. L. hancei has 31 tRNA, while L. balansae contains 33 of these genes. The ML tree (Lithocarpus with L. hancei closely related to L. polystachyus Rehd. The cp genome of L. hancei will help us better construct a robust phylogeny of Lithocarpus and the Fagaceae in the future study.The complete cp genome of  ML tree  fully su


The aim of this article is to review the complex interactions of bacteria with Kaposi’s sarcoma-associated herpesvirus (KSHV) infection and KSHV-induced cancers. KSHV is causally associated with multiple cancers including Kaposi’s sarcoma (KS) and primary effusion lymphoma. Among patients coinfected by HIV and KSHV, patients with KS have a distinct oral microbiome compared to patients without KS. Moreover, KSHV patients have increased levels of salivary bacterial pathogen-associated molecular patterns compared to KSHV-negative patients. KSHV-associated bacterial species can increase KSHV replication and dissemination, and enhance cell proliferation of KSHV-transformed cells. The analysis of bacterial biomarkers associated with KSHV may help improve our understanding of the mechanisms driving KSHV-induced oncogenesis and identify novel targets for improving therapies of KSHV-related cancers.The objective of this article is to review the current status of the bacteria-virus interplay in Kaposi’s sarcoma-associated herpesvirus (KSHV) infection and KSHV-driven cancers. KSHV is the etiological agent of several cancers, including Kaposi’s sarcoma (KS) and primary effusion lymphoma. Due to immunosuppression, patients with KSHV are at an increased risk for bacterial infections. Moreover, among patients coinfected by HIV and KSHV, patients with KS have distinct oral microbiota compared to non-KS patients. Bacterial biomarkers associated with KSHV-driven cancers can provide insights in discerning the mechanisms of KSHV-induced oncogenesis. For example, pathogen-associated molecular patterns and bacterial products of certain bacterial species can regulate the expression of KSHV lytic and latent genes, thereby affecting viral replication and dissemination. In addition, infection with distinct opportunistic bacterial species have been associated with increased cell proliferation and tumorigenesis in KSHV-induced cancers through activation of pro-survival and -mitogenic cell signaling pathways. By elucidating the various mechanisms in which bacteria affect KSHV-associated pathogenesis, we will be able to pinpoint therapeutic targets for KSHV infection and KSHV-related cancers. Fusobacterium nucleatum and Helicobacter pylori have been extensively studied for their carcinogenic effects, as they can produce toxins and effector proteins that induce host cell damage and alter cell signaling pathways involved in cell survival and proliferation [The utilization of bacterial markers to determine cancer pathology has progressed tremendously in recent years. Advances in technology have empowered researchers to more thoroughly elucidate complex interactions between the microbiome and cancer. Microbiome analyses using next generation sequencing report that the colonization of pathogenic bacterial species is increased in cancer patients and exacerbates cancer pathogenesis . Pathogeferation . Contrarferation . Moreoveferation . The anaferation . This reThe human immune system responds to bacterial infection by producing inflammation. Bacteria possess pathogen-associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs) expressed by both non-immune and immune cells ,7. ActivA number of studies have annotated the pathways linking PRR activation and cancer cell proliferation . BacteriHowever, contradictory studies show decreased cell proliferation in cells stimulated with bacterial PAMPs ,23,24. MDue to the substantial evidence of bacterial impact on cancer pathology, it is essential to delineate the intricate interactions of the bacterial microbiome with cancer. The microbiome consists of the microbial community, which maintains intricate interactions. Their effects on the habitat are complex . The humThis review focuses on the effects of bacteria on KSHV infection and KSHV-associated cancers. KSHV is the etiological agent of Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL) . AlthougAs a gammaherpesvirus, KSHV causes life-long persistent infection . The KSHSince not all individuals infected with KSHV develop malignancies, it is especially important to understand the roles co-factors play in the development and severity of KSHV-induced cancers. KSHV most commonly causes cancers in immunocompromised individuals, especially those with HIV infection . PatientFirmicutes and Actinobacteria and decreased levels of the phyla Bacteroidetes and Proteobacterium. Moreover, oral KS patient samples had higher levels of common oral pathogens including bacteria in the genera Corynebacterium and Shuttleworthia [Porphyromonas. gingivalis and Fusobacterium nucleatum [Suppressed immunity associated with HIV increases the risk of opportunistic infections . Severaleworthia . These rucleatum . Moreoveucleatum ,54,55.P. gingivalis, F. nucleatum, Prevotella intermedia, and Streptococcus mutans [P. gingivalis and F. nucleatum medium. The study also examined the mechanism of bacterial-induced reactivation and reported that activation of viral lytic genes by the bacterial mediums occurred through the p38 MAPK pathway as this pathway has been shown to be both essential and sufficient for KSHV lytic replication during primary infection and reactivation [Butyrate, a common metabolic end product of certain bacterial species, has been shown to reactivate herpesviruses including KSHV . Butyrats mutans . Authorstivation ,58,59,60F. nucleatum and P. gingivalis on KSHV reactivation [P. gingivalis and F. nucleatum. Moreover, treatment of BCBL1 cells with different doses of pure individual short chain fatty acids (SCFAs) detected in the mediums of P. gingivalis and F. nucleatum except acetic acid resulted in increased expression of lytic gene RTA (ORF50) [P. gingivalis and F. nucleatum in patients with periodontal diseases compared to healthy controls [P. gingivalis and F. nucleatum had significantly reduced expression of class 1/2 HDACs. In contrast, no effect was seen with Escherichia coli medium [Yu et al. further explored the effects of medium from tivation . Their s (ORF50) . Treatme (ORF50) . Interescontrols . Yu et ai medium . These f2O2) increased KSHV lytic gene expression in HUVEC and PEL cell lines [2O2 causes apoptosis and senescence in primary cells, at lower concentrations, H2O2 can activate multiple redox signaling pathways such as the MAPK pathways [2O2 induction of KSHV reactivation depended on ERK1/2, JNK, and p38 pathways [2O2 can be produced directly by bacteria and indirectly by bacterial stimulation of immune cells, it may be of interest to more thoroughly assess the impact of bacterial infection in causing KSHV reactivation through this mechanism [Besides SCFA production, additional bacterial effects have been linked to KSHV reactivation, including inflammation related to cytokines and ROS . Figure ll lines . Althougpathways . Ye et apathways . As H2O2echanism .S. aureus and P. gingivalis influenced KSHV entry into cells and subsequent expression of viral genes in primary human gingival fibroblasts (HDF) and periodontal ligament fibroblasts (PDLF) [S. aureus induced MAPK-ERK phosphorylation and LPS from P. gingivalis increased NF-κB phosphorylation in HGF [Dai et al. reported that both bacterial PAMPs from s (PDLF) . Dai et s (PDLF) ,64. Theys (PDLF) . These rn in HGF . It has n in HGF ,66. Howen in HGF . It is tS. aureus on KSHV reactivation [S. aureus is a common, gram-negative intracellular pathogen that can colonize in the oral cavity of patients with periodontitis [S. aureus medium, there were increased expression of KSHV lytic genes including RTA, vGPCR (ORF74), ORF-K8.1, and ORF57 [S. aureus medium also induced release of infectious virion particles from both the gingival fibroblasts and PDLF. However, no significant effects from the medium of other gram-positive bacteria tested, such as Bacillus subtilis were observed, suggesting specific effect of the bacterial species. HIV+/KSHV+ patients also had higher levels of salivary LTA, a bacterial ligand for TLR2. However, the LTA ELISA kit used to analyze LTA levels did not distinguish LTA levels among different bacterial species. It would therefore be of interest to examine the effects of LTA from different gram-positive species on KSHV reactivation [Dai et al. also examined the effects of tivation . S. auredontitis . In bothnd ORF57 . Treatmetivation .S. aureus medium reduced the expression of KSHV microRNAs (miRNAs) compared with cells treated with control and fresh medium [S. aureus medium downregulated expression of cyclin D1 and other host proteins responsible for processing cellular miRNAs, including Dicer, and Argonaut 1 and 2. These results suggested that the S. aureus medium might induce KSHV reactivation through the cyclin D1-Dicer-viral miRNAs axis in KSHV-infected oral cell lines [S. aureus and KSHV coinfection since S. aureus is an intracellular pathogen that can invade and survive in many cells including fibroblasts and endothelial cells. The authors concluded that coinfection in the same single cells induced KSHV reactivation. Moreover, a bacterial analysis of saliva samples in HIV+ patients showed 72% S. aureus compared to 37% in HIV-subjects [S. aureus (MRSA) infection is a common complication in HIV patients [S. aureus, it may be worthwhile to investigate the effects of S. aureus in additional sites other than the saliva in HIV/KSHV-coinfected patients. Moreover, this study examined the conditioned medium of S. aureus which might contain many bacterial products besides LTA. It may be important to study the effects of purified LTA on KSHV-infected cells.Mechanistically, Dai et al. found that h medium . KSHV mih medium . Furtherll lines . The stusubjects . This stpatients . Due to P. gingivalis and KSHV infection in HIV+ patients, and found that 11.3% were positive for both P. gingivalis (a periodontitis-associated bacterium) and KSHV [P. gingivalis medium and LPS increased the expression of KSHV lytic genes in latently infected primary human oral fibroblasts through TLR4 [P. gingivalis [P. gingivalis medium and LPS activated the MAPK pathways in KSHV-infected oral cells by inducing phosphorylation of p38 and JNK. However, treatment with p38 or JNK inhibitors did not completely hinder viral lytic reactivation, implying other pathways may be involved [E. coli [In a separate study, Dai et al. analyzed saliva samples for the presence of and KSHV ,70. Therand KSHV . Mechaniugh TLR4 . Howeverngivalis . Likewisinvolved . Interes[E. coli . The dif[E. coli .While lytic activation of KSHV is necessary for disseminating the infection, latency is essential for establishing persistent KSHV infection . Upon KSGruffaz et al. analyzed the expression of TLRs in rat primary embryonic metanephric mesenchymal precursor (MM) cells and KSHV-transformed MM (KMM) cells . MultiplPseudomonas aeruginosa [P. aeruginosa is a gram-negative opportunistic pathogen that can infect immunosuppressed individuals [P. aeruginosa infection resulted in increased proliferation in KSHV-infected BJAB and KMM cells but had no significant effects in the KSHV uninfected control cells [P. aeruginosa stimulation increased cell proliferation and efficiency of colony formation in soft agar of KMM cells but had no significant effect on the untransformed MM cells [P. aeruginosa increased inflammatory cytokines and activation of p38, ERK1/2, and JNK MAPK pathways in KMM cells through LPS and flagellin ligands [2O2, SCFAs, quorum signaling molecules, etc.) that may induce proliferation in KSHV-latently infected cells, and therefore may be worth further investigation.Markazi et al. further investigated the effects of TLR4 by stimulating MM/KMM and BJAB/KSHV-infected BJAB cells (EBV-negative B-cell lymphoma cells) with ruginosa . P. aeruividuals . P. aeruol cells . As KMM ol cells , MarkaziMM cells . Mechani ligands . These rF. nucleatum and P. gingivalis produce SCFAs which suppress HDAC activity, resulting in increased hyperacetylation and subsequent expression of lytic gene expression [The collection of studies reviewed herein has demonstrated that bacteria can affect KSHV-induced cancers by numerous mechanisms, including through bacterial PAMPS and bacterial-secreted byproducts. Indeed, microbiome analyses reveal increased levels of pathogenic bacteria and secreted PAMPs in immunosuppressed HIV/KSHV-coinfected patients ,54,67. Bpression ,55,56. Bpression ,64,67. Kpression . Moreovepression ,78. Analpression . By elucSince interactions among bacterial species and the host are complex, it is crucial to more thoroughly investigate the microbial signature of HIV/KSHV-coinfected patients to enhance our understanding of bacterial biomarkers and the underlying mechanisms that contribute to KSHV pathology. Many studies have reported that immunosuppression and inflammation drive KSHV pathology, both of which are applicable to bacterial infection . InteresWhile the majority of microbiome analyses in HIV/KSHV-coinfected patients used saliva samples, it may be beneficial to assess the microbiome in additional sites, such as KS lesions present on different organs  as microbial species vary depending where they are located . MoreoveIt has been demonstrated that the NF-κB, STAT3, and MAPK pathways play crucial roles in KSHV reactivation and cell proliferation of KSHV latently infected cells ,63,77,78The utilization of KSHV-infected cells in cell culture experiments has been proven invaluable for providing data on the effects of bacteria on KS pathology. The KSHV-infected cells used in both the Gruffaz et al. and Markazi et al. studies were developed by infecting primary rat embryonic metanephric mesenchymal precursor cells ,78,80. TFirmicutes phylum in KS patient, Firmicutes bacteria are potent producers of SCFA such as butyrate, which can reactivate KSHV and other herpesviruses [Firmicutes bacteria potentially complicates the use of probiotic bacteria such as Lactobacillus as “anti-inflammatory” bacteria as they can be potent producers of SCFAs [The collection of studies explored in this review focus predominantly on pathogenic bacteria enhancing KSHV-induced cancer pathology. However, bacteria explored as anti-cancer therapies have also been of interest for multiple cancer types ,26. Moresviruses ,55,86. Tof SCFAs . FurtherNotwithstanding the potential of anti-tumor bacterial species, the studies explored in this review strongly emphasize the negative effects of pathogenic, opportunistic infection in patients with KSHV-induced cancers. As these cancers continue to be the most common cancer types in immunosuppressed patients despite increased use of antiretroviral drugs, it is evident that more efficient treatments are in demand . Microbi