Heterobasidion parviporum Niemelä & Korhonen is an economically important basidiomycete, causing root and stem rot disease of Norway spruce (Picea abies (L.) Karst) in Northern Europe. The H. parviporum genome encodes numerous small secreted proteins, which might be of importance for interacting with mycorrhiza symbionts, endophytes, and other saprotrophs. We hypothesized that small secreted proteins from H. parviporum (HpSSPs) are involved in interspecific fungal interaction. To identify HpSSP-coding genes potentially involved, we screened the H. parviporum effectome and compared their transcriptomic profiles during fungal development and in planta tree infection. We further conducted phylogenetic analysis, and identified a subset of hypothetical proteins with nonpredicted domain or unknown function as HpSSPs candidates for further characterization. The HpSSPs candidates were selected based on high-quality sequence, cysteine residue frequency, protein size, and in planta expression. We subsequently explored their roles during in vitro interaction in paired cultures of H. parviporum with ectomycorrhizal Cortinarius gentilis, endophytic Phialocephala sphaeroides, saprotrophs , respectively. The transcriptomic profile revealed that a large proportion of effector candidates was either barely expressed or highly expressed under all growth conditions. In vitro dual-culture test showed that P. sphaeroides and C. gentilis were overgrown by H. parviporum. The barrage zone formation or no physical contact observed in paired cultures with the saprotrophs suggest they had either combative interaction or antibiosis effect with H. parviporum. Several HpSSPs individuals were up- or downregulated during the nonself interactions. The results of HpSSPs gene expression patterns provide additional insights into the diverse roles of SSPs in tree infection and interspecific fungal interactions. Heterobasidion parviporum Niemelä & Korhonen is one of the most severe root rot pathogens colonizing Norway spruce (Picea abies (L.) Karst). H. parviporum root rot is common in southern Finland with annual economic losses estimated at 40 million euros for the Finnish forest industry [Phlebiopsis gigantea (Fr.) Jül shares the same ecological niche with H. annosum sensu lato (s.l.) by rapidly colonizing the stump surface for space and nutrients, which makes it an ideal biocontrol agent against Heterobasidion species [P. gigantea, other saprotrophic fungi, mycorrhiza, and endophytes are also important sources of potential biocontrol agents. Many previous studies have focused on searching for new biocontrol agents, but very few investigated the role of small secreted proteins in influencing the outcome of the interaction.The basidiomycete industry ,2. Freshindustry . Typical species ,4. ApartTrametes versicolor and Trichoderma harzianum [T. versicolor during interspecific mycelial interaction showed expression changes in genes related to cell wall biosynthesis, cell division, carbohydrate and nitrogen metabolism, glycine-rich RNA binding protein, playing wide roles in hyphal growth and stress response [Fungal competition in wood is commonly considered as either primary resource capture or secondary resource capture . Primaryarzianum . Nonselfarzianum . Such moresponse . HoweverH. parviporum revealed that around 7% of genes were predicted to be secreted proteins , known as the secretome [H. parviporum, and selected high-priority candidates, named H. parviporum small secreted proteins (HpSSPs), from the predicted effectome based on protein size and the frequency of cysteine residues, or the upregulation profiles in planta. We hypothesized that HpSSPs would be involved in interspecific fungal interaction. The aims of the present study were: (1) characterization of transcriptomic profiles of HpSSPs during fungal development (conidiospores and free-living mycelia growth) and tree infection (saprotrophic and necrotrophic growth); (2) the modulation of a set of HpSSPs expression over the course of fungal interactions between H. parviporum and endophytic, mycorrhizal and other saprotrophic fungi in artificial media.The availability of genome sequences of fungal pathogens has enabled the analysis of the expression level of numerous genes under diverse experimental conditions. The recent genome analysis of ecretome . A smallecretome . Effectoecretome , with reecretome ,17. Howeecretome . In thisHeterobasidion parviporum 96026 HAMBI 2359, Phialocephala sphaeroides strain 222, Stereum sanguinolentum FBCC 1148, Phlebiopsis gigantea (courtesy of Kari Korhonen), Phanerochaete chrysosporium HE-446 FBCC 280, Mycena sp. JH226, Cortinarius gentilis K94 FBCC 546) were maintained on malt extract agar (MEA) . Homokaryotic H. parviporum strain 96026  was provided by courtesy of Kari Korhonen. P. sphaeroides in this study, originally isolated from roots of Norway spruce from minerotrophic pristine mire (Vaccinium myrtillus spruce swamp (MK) [P. sphaeroides), saprotrophic fungi , the ectomycorrhizal fungus (C. gentilis) according to their lifestyles. Each fungal species was cocultured with H. parviporum on MEA in a 90 mm diameter petri dish. Two agar plugs (5 mm diameter) were placed at a distance of 60 mm on MEA surface preoverlaid with a piece of cellophane membrane. H. parviporum self-pairing (HpHp) was used as an additional control. The fungi were grown at 20 °C in darkness. Mycelia from the paired cultures were collected at different interaction stage, including precontact (t1), initial contact (t2) , and days postcontact (t3) . There were three biological replicates at each interaction stage, with each replicate consisting of mycelia from different MEA plates. Total RNA was isolated by using TRI reagent (Sigma Aldrich), following the manufacturer’s instructions [All fungal isolates (amp (MK) , is a daH. parviporum 96026 was used to identify the effectome. The genome contains approximately 10,500 predicted genes, 287 scaffolds, and 759 secreted proteins [The sequenced genome of proteins . Subsequproteins  and LOCAWe filtered out a small number of candidates with incorrect gene structures . Twelve small secreted proteins (HpSSPs) were selected from the remaining effectome based on the length of amino acids, the percentage of cysteine residues, or expression patterns at diverse developmental stages.H. parviporum effector candidates was monitored during developmental stages of the fungus: conidiospores, free-living mycelia, saprotrophic, and necrotrophic growth. The methods of sample preparation and collection were as previously described [H. parviporum within the inoculated trees during in planta growth, whereas samples for necrotrophic growth were obtained from necrotic lesions surrounding living cells of the inoculated trees after three and half months [Expression of escribed . Brieflyf months .18 Primer  was mixed with the DNA-free RNA template at 65 °C for 5 min for primer annealing and then incubated on ice. After annealing, the reverse transcriptase and components including buffer, dNTPs, and RNase inhibitor were added. The reaction mixture was incubated at 42 °C for 60 min and the reaction was stopped by heating at 70 °C for 5 min.Complementary DNA (cDNA) was synthesized according to the Thermo-Fisher Scientific’s protocol. We added 1 µL of DNase I  to 1 µg of total RNA to remove genomic DNA and subsequently deactivated by heating at 65 °C for 10 min in the presence of 1 µL of 50 mM EDTA. A total of 100 µM Oligo(dT)https://lifescience.roche.com/en_fi/brands/universal-probe-library.html#assay-design-center). The cDNA template (3 µL) was diluted into 97 µL of nuclear-free water. The total qPCR reaction volume was 15 µL, including 5.5 µL of the diluted cDNA, 1 μL of forward primer (10 μm), 1 μL of reverse primer (10 μm), and 7.5 μL of two times LightCycler 480 SYBR Green I Master . The reaction program was as follows: 5 min denaturation at 95 °C, 45 amplification cycles of 10 s at 95 °C, 10 s annealing at 60 °C, and extension 20 s at 72 °C. The relative quantities of HpSSP-coding genes were normalized with four housekeeper genes in H. parviporum, including RNA polymerase III transcription factor (RNA Pol3 TF), mannosyltransferase (Maan transf), tryptophan catabolism (Tryp metab), ribosomal protein S23 (RiboS23). All primers used in this study are shown in We designed the primers for qPCR at Roche Universal Probe Library Assay Design Center  contained less than 300 aa, with the largest group containing 100–200 aa (49 candidates), followed by those between 200 aa and 300 aa (39 candidates). The number of proteins with the size between 300 to 400 aa was approximately equal to those of size 400–500 aa, 18 and 17 candidates, respectively. Only a small number of proteins (24 candidates) had the length of 500–1000 aa, making up 14.7%. Only seven candidates had over 1000 aa, as seen in H. parviporum (HpSSPs). Their protein size, the cysteine residue frequency, and the homologs H. irregulare are shown in H. irregulare, as seen in Since a large proportion of the effector candidates consisted of hypothetical proteins with unknown function, phylogenetic analysis was utilized to explore the potential functional group of the hypothetical proteins based on their sequence similarity to proteins of known function. The phylogenetic analysis showed that 163 effector candidates were distinctly divided into four main groups, as seen in With the sequenced RNAs from free-living mycelia (MYCEL), conidiospores (SPORE), saprotrophic sawdust growth (SAP), and necrotrophic growth in the necrotic stem tissue (NECT) in a previous study , we inveWe closely inspected whether the selected HpSSP-coding genes were significantly expressed in NECT and SAP compared to SPORE. HpSSP1.244, as a predicted effector, as seen in P. chrysosporium and P. gigantea grew much faster, such that the initial contact with H. parviporum was earlier than for the other tested fungi. H. parviporum grew much faster than C. gentilis and P. sphaeroides, accumulating much more biomass. The time of precontact (t1) and initial contact (t2) in self-interaction was at 8 days postinoculation (dpi). The mycelia of H. parviporum self-pairing (HpHp) postcontact (t3) was collected at 19 dpi, as seen in H. parviporum and C. gentilis (HpCg), mycelia at t1 and t2 were harvested at 12 dpi, and the mycelia at t3 were collected at 16 dpi, as seen in The interaction outcome was not only dependent on the species combinations, but also partly on the growth rate of the fungal isolates. This led to differences in the time points at precontact (t1), initial contact (t2), and postcontact (t3). gentilis and P. sphaeroides, which developed at much slower speed than H. parviporum, were easily overgrown by the conifer pathogen, as seen in H. parviporum vs. P. chrysosporium (HpPc), H. parviporum vs. P. gigantea (HpPg), and H. parviporum vs. S. sanguinolentum (HpSs), as seen in H. parviporum produced mycelia that partly invaded into the territory occupied by P. chrysosporium, as seen in H. parviporum and Mycena sp. (HpMy). Antagonism or antibiosis between fungi without hyphal contact can be mediated via volatile or diffusible chemicals or other antifungal metabolites [P. chrysosporium, P. gigantea, S. sanguinolentum, and Mycena sp. might have a competitive impact on the growth of H. parviporum.The self-pairing produced uniform mycelial mats, and no barrage zone was formed, as seen in abolites . BarrageTo investigate the gene expression pattern of the selected HpSSPs responsive to the tested endophyte, ectomycorrhiza, and saprotrophic basidiomycetes, the normalized relative quantities of the 12 selected HpSSP-coding genes were assessed at the precontact (t1), initial contact (t2), and postcontact (t3) during the interspecific fungal interactions. When the HpSSPs expression profiles of each pairwise combination were clustered, HpSSP2.330, HpSSP2.152, and HpSSP35.8 were upregulated during the nonself interactions, except in HpMy, while HpSSP26.11, HpSSP3.169, and HpSSP3.534 were commonly downregulated, especially at t3, as seen in H. parviporum and the endophyte P. sphaeroides (HpPs), we found strong regulation of the commonly expressed HpSSP individuals, compared to the combination with other wood-decay basidiomycetes, as seen in C. gentilis was also replaced by the pathogen on malt extract agar, and HpCg likely shared the similar expression dynamics to HpPs, including the commonly upregulated ones , HpSSP26.11, HpSSP33.48, and HpSSP33.91, as seen in During the interaction between H. parviporum were also investigated in pairwise combinations with three wood-decaying basidiomycetes. Dense mycelia were formed at the interaction zone in H. parviporum vs. P. chrysosporium (HpPc), as seen in H. parviporum vs. P. gigantea (HpPg), as seen in H. parviporum vs. S. sanguinolentum (HpSs), as seen in P. chrysosporium and P. gigantea should be more aggressive against H. parviporum based on the less biomass produced by H. parviporum. Unlike in HpPs and HpCg, the expression of HpSSP33.48 in HpPg and HpPc was upregulated over the observed period, as seen in H. parviporum and saprotrophic Mycena sp. (HpMy) was antagonism at a distance, as seen in Interactions with white-rot H. parviporum, as a necrotrophic fungal pathogen of Norway spruce, has intimate interactions with other fungal species, such as endophytes and mycorrhiza during invasive growth. In our pairwise combinations, hyphal intermingling was observed in self-pairings, which may result from cooperative or neutral interaction [H. parviporum overgrew endophytic P. sphaeroides and mycorrhizal C. gentilis in this study. However, the root endophyte P. sphaeroides was considered to possess inhibitory capability to reduce the growth of H. parviporum [H. parviporum overwhelmed the mycelial growth of P. sphaeroides, which might have affected its inhibitory effect. A barrage zone was formed when H. parviporum was paired with P. chrysosporium, P. gigantea [S. sanguinolentum. The barrage zone formation in HpPg was temporary, as overgrowth of H. parviporum by P. gigantea was observed during prolonged incubation. Mycelial growth of H. parviporum encountered an antibiosis effect when paired with Mycena sp., possibly due in part to the effects of diffusible chemical production [H. parviporum genome sequences has provided a much better understanding of the regulation of small secreted proteins (HpSSP) possibly involved in pathogenicity, as well as in the fungal interaction under those pairwise combinations. We found that a subset of HpSSPs shared similar expression dynamics over interaction combinations, while some HpSSPs showed combination-dependent expression. Although the identity of the HpSSPs documented in our study is still unknown, this does not diminish their functional relevance.Interspecific fungal interaction, as a key feature of fungal community formation, dramatically affects the size of the populations and the fitness of individuals. Fungal competition is commonly applied in screening for potential biocontrol agents against phytopathogens of trees. eraction . H. parvrviporum . The domgigantea , and S. oduction . BarrageH. parviporum was predicted to have 21.4% HpSSPs in its secretome (163/759). This percentage is greater than the proportions of effectors in the secretomes of other white rot fungi, such as T. versicolor (13.1%), Dichomitus squalens (11.2%), and Stereum hirsutum (8.5%) [H. parviporum effector candidates during fungal growth in wood sawdust (SAP), fungal invasion in necrotic stem tissues (NECT) and the profile in conidiospores (SPORE), and mycelial growth on artificial media (MYCEL). The results showed a large proportion of effector candidates were either barely expressed or highly expressed under all growth conditions. A few candidates were upregulated in both NECT and SAP relative to SPORE and MYCEL, including HpSSP3.169 and HpSSP1.590. No NECT-specific gene was observed in the transcriptome analysis. Effector proteins are believed to be temporally expressed, with a set of effectors highly expressed during the early infection and some highly expressed at the later stages [Diverse strategies utilized by fungal individuals might confer flexibility towards local changes in the environment to increase their fitness, which may be partly derived from elaborately regulated gene expression profiles in response to diverse conditions . Fungi hm (8.5%) . Here, wr stages ,31. In oH. annosum, also had a high transcript level in both saprotrophic and necrotrophic conditions, as well as in liquid media [H. annosum, showed low transcript level in necrotrophic conditions, but high transcript level in saprotrophic growth in wood [Nicotiana benthamiana [Postia placenta grown on cellulose was distinct compared to growth on glucose [Although HpSSP3.169 and HpSSP1.590 were predicted noneffectors, they were highly expressed in SAP and NECT. Their roles in pathogenicity during host–pathogen interaction need to be further explored. HpSSP1.244 showed considerably high induction in SPORE, MYCEL, SAP, and NECT, which indicated that it might contribute to all lifestyles of the pathogen. HaSSP1, a homolog of HpSSP1.244 in id media . HpSSP3. in wood . HpSSP35thamiana . The expthamiana ,34. The H. parviporum during interspecific interaction has been poorly documented thus far [H. parviporum responded with both common and combination-dependent HpSSP regulation when interacting with different fungal species. A mycoparasitic fungus, Clonostachys rosea, responded with both common and specific gene expression during interaction with the plant pathogen Botrytis cinerea and Fusarium graminearum. The common responses in C. rosea included sugar and small organic compound transporters potentially involved in nutrient uptake. The specific responses against the fungal pathogens were dominated by genes associated with membrane transport, biosynthesis of secondary metabolites, and carbohydrate degradation [H. parviporum and P. gigantea has been widely studied, since P. gigantea is commonly used as a biocontrol agent because of its competitive advantage against H. parviporum [H. annosum in contact with the hyphae of P. gigantea has been documented in dual cultures in agar medium [H. annosum genes involved in glucose metabolism transport, signal transduction, and defense were differentially downregulated after exposure to P. gigantea culture filtrate. This might indicate that the nutrient processing, acquisition, and resistance machinery of H. annosum were suppressed by P. gigantea during antagonistic interaction [The regulation of small secreted proteins (HpSSP) from the forest pathogen thus far ,36,37. Hradation . During radation . After eradation . In the rviporum . Hyphal r medium . A numbe


Chlamydia trachomatis serovars are obligate intracellular bacterial pathogens mainly causing ocular and urogenital infections that affect millions of people worldwide and which can lead to blindness or sterility. They reside and multiply intracellularly within a membrane-bound vacuolar compartment, known as inclusion, and are characterized by a developmental cycle involving two morphologically and physiologically distinct chlamydial forms. Completion of the developmental cycle involves the secretion of > 70 C. trachomatis proteins that function in the host cell cytoplasm and nucleus, in the inclusion membrane and lumen, and in the extracellular milieu. These proteins can, for example, interfere with the host cell cytoskeleton, vesicular and non-vesicular transport, metabolism, and immune signalling. Generally, this promotes C. trachomatis invasion into, and escape from, host cells, the acquisition of nutrients by the chlamydiae, and evasion of cell-autonomous, humoral and cellular innate immunity. Here, we present an in-depth review on the current knowledge and outstanding questions about these C. trachomatis secreted proteins. Chlamydia trachomatis serovars are human pathogens causing mostly ocular and genital infections [C. trachomatis strains comprise three biovars, which can be further divided into 15 main serovars, based on antigenic variation of the major outer membrane protein (MOMP): the trachoma biovar (serovars A-C); the genital biovar (serovars D-K); and the lymphogranuloma venereum (LGV) biovar (serovars L1-L3). Most C. trachomatis infections are caused by genital strains, but studies on host cell-C. trachomatis interactions are usually performed with a prototype serovar L2 strain.fections , 2. ThesC. trachomatis is member of a Phylum (Chlamydiae) of Gram-negative bacteria, comprising one Class (Chlamydiia) and one Order , characterised by obligate growth within eukaryotic cells and including species that infect vertebrates, invertebrates, and eukaryotic microorganisms such as amoeba [Chlamydiales, the Chlamydiacea Family currently consists of 16 Chlamydia species [C. trachomatis. In addition, the Chlamydiacea comprises C. pneumoniae, causing pulmonary infections in humans, and pathogens of a wide range of non-human vertebrates. Among the latter, C. abortus, C. caviae, C. felis, and C. psittacci have zoonotic potential.s amoeba . Among t species , includiChlamydiales are characterized by a developmental cycle involving two distinct morphological forms, the small, infectious and non-replicative, elementary bodies , and the larger, non-infectious and replicative, reticulate bodies  , which after release from the host cell can infect neighbouring cells .The iewed in , 6). ThiC. trachomatis interferes with a wide range of host cell processes during its developmental cycle . Subversion of host receptor-mediated signalling and of the actin cytoskeleton and its key regulators promotes chlamydial adherence and invasion of host cells. While intracellularly, C. trachomatis reshapes the protein and lipid composition of the inclusion membrane by selective interactions with molecules controlling and mediating host cell vesicular trafficking, segregating from the endolysosomal pathway and forming a unique and stable vacuolar compartment that localizes near the centrosome. The interaction with host cell vesicular and non-vesicular transport pathways also enables C. trachomatis to acquire nutrients and lipids required for its growth. In addition, among other aspects, intravacuolar C. trachomatis modulates host cell survival and death and the innate immune signalling. Finally, to exit from the host cell, C. trachomatis subverts the host cell cytoskeleton and calcium-signalling.iewed in ; Figure C. trachomatis involves the timely secretion of many chlamydial proteins. We will survey and discuss the knowledge on the identity and function of C. trachomatis secreted proteins that participate or might participate in the subversion of host cell processes. This knowledge has significantly increased in recent years because of developments in methods to genetically manipulate C. trachomatis [C. trachomatis [Completion of the developmental cycle and subversion of host cells processes by chomatis –15 (and chomatis ) that fochomatis .C. trachomatis, its genome encodes the Sec system, a type II secretion (T2S) system, a type III secretion (T3S) system, Sec-exported polymorphic membrane proteins (Pmps) containing type V secretion (T5S) system/autotransporter signals, and several other outer membrane proteins [. The Sec- and T3S system-dependent transport of C. trachomatis proteins by heterologous bacteria [C. trachomatis mutants in the T2S system-associated ATPase [C. trachomatis developmental cycle and for Chlamydia-host cell interactions. All together, these systems enable the delivery of chlamydial proteins to different aqueous and membranaceous compartments within bacterial and mammalian host cells . The use in C. trachomatis-infected cells of small molecules reported to inhibit the Sec system and the T3S system also initially indicated the importance of these protein transport systems for chlamydiae [C. trachomatis are questionable.Gram-negative bacteria use several protein transport systems that are essential for interactions with other cells and with the extracellular environment –20. In tproteins , 22  [C. trachomatis with host cells [C. trachomatis proteins: (i) that localize in the inclusion membrane, functioning as effectors mediating the chlamydiae-host cell interaction; (ii) that localize and function as effectors in the host cell plasma membrane, cytoplasm or nucleus; (iii) that localize in the inclusion lumen, where they function and/or from where they might be transported into the inclusion membrane and/or host cell cytosol, or are released in the extracellular environment after chlamydial exit.Chlamydial outer membrane proteins, such as the Pmps , outer m (Ctad1)  are impost cells , but theChlamydia proteins mediating bacterial-host cell interaction are the inclusion membrane proteins (Incs) [Chlamydia species are mostly unrelated to each other [C. trachomatis, and several more might exist [Chlamydiales, including endosymbionts of free-living amoeba [The most prominent group of s (Incs) , 41. Altch other , 43, thech other , 41. Thiht exist , 43. Othg amoeba , also lig amoeba , 43.C. psittaci or immunized with killed EBs, proteins absent in purified EBs or in uninfected cells were localized at the inclusion membrane by immunofluorescence (IF) microscopy [C. psittaci DNA, and this led to the identification of a gene encoding an inclusion membrane protein, named IncA [C. psittaci IncB and IncC [C. trachomatis, released in 1998 [C. trachomatis [C. trachomatis led to the identification of Incs D, E, F, and G [The first report of the identification of an Inc dates to 1995 . By usincroscopy . The sammed IncA . A similand IncC . Data fr in 1998 , reveale in 1998 , 48, whichomatis . MoreoveF, and G .C. trachomatis for genes that could encode additional Incs [C. trachomatis Incs and showing by IF microscopy that they localize at the inclusion membrane, the bilobed hydrophobic region was defined as a characteristic motif of Incs and a determinant of their localization [C. trachomatis Incs [C. trachomatis [After the identification of Incs A, B and C, the characteristic bilobed hydrophobic region was used to search the genome of nal Incs . By raislization . In substis Incs , 52–57, tis Incs , 43. Thechomatis , 17, 58,chomatis , but it chomatis . DiffereChlamydia [Chlamydia were unavailable at the time, but it had been shown that chlamydial proteins could be type III secreted by heterologous bacteria [Shigella flexneri [C. trachomatis Inc proteins were confirmed as T3S substrates [The lack of a cleavable Sec signal peptide on the first identified Incs and the discovery of homologues of T3S system genes in hlamydia , 60, sughlamydia , 51, 61.bacteria . Using tflexneri , 63, whibstrates , 59, 64.C. trachomatis gene expression revealed at least three classes of Incs depending on their corresponding messenger RNA (mRNA) levels during the chlamydial developmental cycle: early-cycle Incs (highest mRNA levels between ~2-6 h post-infection); mid-cycle Incs (highest mRNA levels between 6-20 h post-infection); late-cycle Incs (highest mRNA levels after ~20 h post-infection) [Analyses of fection) , 64–66. C. trachomatis manipulates host cell vesicular trafficking at least to avoid the phagolysosomal route leading to bacterial destruction and to intercept vesicles containing nutrients required for bacterial growth and inclusion expansion [C. trachomatis Incs ; Table 1) have been shown to be directly involved .As an intravacuolar pathogen, xpansion . Given tC. trachomatis normally resides and multiplies within a single inclusion containing several chlamydiae. In contrast, C. trachomatis-infected cells microinjected with anti-IncA antibodies show multiple inclusions within a single host cell [C. trachomatis clinical isolates residing in multiple inclusions within infected cells have mutations in incA and lack the IncA protein in the inclusion membrane [, which was confirmed when C. trachomatis incA insertional mutants were generated and characterized [ost cell . Furthermembrane , 70. Thicterized , 71.. However, the recent determination by X-ray crystallography of the three-dimensional (3D) structure of almost all the cytosolic region of IncA [In eukaryotic cells, membrane fusion requires interactions between soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in opposing lipid bilayers . Bioinfo of IncA , indicat of IncA , 79.C. trachomatis IncA can also interact with mammalian SNAREs (vesicle-associated membrane proteins (VAMPs) 3, 7, and 8), which are recruited to the periphery of the inclu-sion [C. trachomatis lacking IncA [.clu-sion . The inting IncA . Moreoveing IncA , 80. Thi. Because incA is expressed at mid-cycle [C. trachomatis isolates with incA mutations results in milder symptoms and reduced bacterial load [incA mutation revealed slower growth in cultured cells [C. trachomatis incA null-mutant strain reveals no growth defect in cultured cells [C. trachomatis proteins that should also contribute to inhibition of fusion with endocytic vesicles.In summary, IncA mediates inclusion fusion and has the capacity to inhibit endocytic membrane fusion id-cycle , 64, thiial load . Moreoveed cells . Howevered cells . This sutrans-Golgi network (TGN) [C. trachomatis inclusions and a screen for human proteins interacting with C. trachomatis Incs both disclosed the importance of the retromer in chlamydial host cell infections [. The IncE:SNX5 interaction has been studied at a structural level [C. trachomatis [C. trachomatis infection and that IncE might subvert retromer and lysosomal function by binding SNXs 5 and 6. This IncE-dependent subversion of the retromer might, for example, enable C. trachomatis to acquire nutrients, or to avoid lysosome- or autophagy-mediated killing [The retromer is a protein complex, including at least one sorting nexin (SNX), which recycles cargo from endosomes to the plasma membrane or to the rk (TGN) . Proteomfections , 85. Sevfections , and Incal level –88. Thischomatis –88. M6PRchomatis . As deplchomatis , 85, thi killing .C. trachomatis inclusion membrane [C. trachomatis protein, Inc CpoS , has been shown to bind and modulate the function of RABs .RAB GTPases are master regulators of eukaryotic vesicular trafficking , and sevmembrane –96. DespC. trachomatis encoding epitope-tagged CpoS, revealed or confirmed interactions of this Inc with RABs 1, 2, 4, 6, 8, 10, 14, 18, 33, 34, and 35 [C. trachomatis cpoS null mutants [Yeast two-hybrid and pull-down experiments first revealed an interaction between CpoS and RAB4 . More re, and 35 , 96, 98. mutants , 96, 99.. Furthermore, this accumulation of transferrin is dependent on RABs 4 and 35 [C. trachomatis-infected cells. Accordingly, cpoS C. trachomatis mutants are attenuated in the generation of infectious progeny in cultured cells and in mice infection models [Expression of CpoS is toxic to yeast cells, which led to the identification of proteins related with clathrin-coated vesicles that when overexpressed suppressed CpoS-mediated toxicity . Somewha4 and 35 . In summn models , 100. ThC. trachomatis Incs CpoS, IncC and CT383  have been reported to control these processes in Chlamydia-infected cells.Intracellular pathogens must ensure the integrity of their replicative niche and therefore they often inhibit host cell death . IntravaC. trachomatis cpoS null mutants die much more frequently than cells infected by the wild-type strain [C. trachomatis [CpoS derives its name from the observation that cells infected by e strain . This hachomatis , 100.et al. performed insertional mutagenesis of eleven inc genes eventually revealing that infection by C. trachomatis cpoS, incC, or ct383 null mutants resulted in increased host cell death [cpoS mutant [incC and ct383 mutants were also defective for generation of infectious progeny in cultured cells and attenuated in a mouse infection model [inc mutants revealed both multiple inclusions per infected cell and premature lysis of the inclusion membrane, the increased cytotoxicity was proposed to be a consequence of the release of the chlamydiae in the host cell cytosol [inc mutant chlamydiae leads to autophagy-dependent host cell death [In one study, Weber ll death . As the S mutant , 100, thet al. [cpoS mutant was shown to activate the stimulator of interferon genes (STING), leading to its re-localization from the endoplasmic reticulum (ER) to perinuclear vesicles and to the triggering of a signalling pathway that results in an enhanced interferon (IFN) response [cpoS mutant C. trachomatis was reduced in STING-deficient cells, this reduction was not observed upon pharmacological inhibition of the transport of STING from the ER into perinuclear vesicles or of the downstream signalling pathway [C. trachomatis are independent processes [C. trachomatis could be related with control of calcium pools in the ER [In the study by Sixt et al. , infectiresponse . While h pathway . This inrocesses . Experimn the ER .In summary, in one model, lack of CpoS leads to inclusion lysis and autophagy-dependent host cell death , while iC. trachomatis manipulates and remodels the eukaryotic cytoskeleton  at different stages of the developmental cycle [C. trachomatis Incs IPAM (inclusion protein acting on microtubules) and InaC (inclusion membrane protein for actin assembly)  have been shown to be involved in the subversion of microtubules and microfilaments and in Golgi redistribution.al cycle –110, andal cycle . Thus faC. trachomatis inclusion, which suggested the involvement of an Inc [.Host cell microtubules accumulate in a nest-like structure around the f an Inc . IPAM waf an Inc . In infef an Inc , 109. InC. trachomatis strains was used to screen for the chlamydial genes involved in this F-actin accumulation [. Although experiments with drugs interfering with actin polymerization suggested that InaC-dependent remodelling of F-actin around the inclusion could be required for Golgi redistribution [C. trachomatis strain overexpressing InaC indicated that these are two independent processes [Filamentous (F)-actin also accumulates around the inclusion . A collemulation . This lemulation , which wmulation   are small GTPases regulating vesicular trafficking, actin remodelling and the structure of the Golgi complex , and thenclusion , 112. Monclusion , 112  are involved.Eukaryotic organelles can interact through membrane contact sites (MCSs), corresponding to areas of close apposition between membranes involving tethering and functional protein complexes, but where membrane fusion does not occur . Besides. A small-interfering RNA (siRNA) screen [C. trachomatis growth [In mammalian cells, the transport of ceramide from the ER to the Golgi involves an MCS and is mediated by the ceramide transporter (CERT); ceramide is then converted into sphingomyelin by synthases in the Golgi   screen , and the) screen , 122, bos growth , 122.CERT, its ER binding partners (VAMP-associated protein A (VAPA) and VAPB), and host cell sphingomyelin synthases were shown to localize near the inclusion membrane by IF microscopy , 122. ImC. trachomatis and ectopically expressing epitope-tagged CERT revealed an interaction with IncD that was further validated [C. trachomatis overexpressing IncD [. In the inclusion, ceramide should be converted into sphingomyelin by a sphingomyelin synthase recruited to the inclusion membrane through a currently unknown mechanism [How is CERT recruited to the inclusion membrane? Immunoprecipitation from extracts of cells infected by alidated . Furthering IncD . Recruiting IncD . In summechanism .C. trachomatis Inc (IncV) is a factor establishing ER-inclusion tethering [C. trachomatis Incs [C. trachomatis overexpressing IncV, and slightly reduced in cells infected by a C. trachomatis incV mutant [. A C. trachomatis incV mutant does not display a growth defect in cultured cells [Another ethering . An intetis Incs . The Inctis Incs . RecruitV mutant . DiffereV mutant  , CT222, IPAM, CT224, CT228, IncB, IncC, CT288, and CT850; Table 1) have been shown to concentrate at regions of the inclusion membrane near the centrosome, known as inclusion microdomains, which are also enriched in cholesterol and in the phosphorylated active form of Src family kinases [Chlamydia species [About 2 h after invasion of host cells by in motor , 127. Se kinases . These k species . The str species , and has species . This su species , the ER- species , 125, an species , could c species , a possiC. trachomatis IncB, its orthologue from C. psittaci can bind synaptosome-associated protein (SNAP)-associated protein (SNAPIN) and this might enable the inclusion to associate with microtubules [Based on the functions of the associated Incs, the inclusion microdomains have been described as platforms for interaction with the centrosome, microtubules and the actin-myosin cytoskeleton , 132. Asotubules . Inc CT2otubules ; CCDC146otubules . Inc CT8otubules , which i. C. trachomatis Inc proteins MrcA and CT228  have been shown to regulate extrusion.Chlamydial release from infected cells can occur by host cell lysis (preceded by permeabilization of the inclusion membrane) or extrusion (in which the entire inclusion is ejected from the host cell)   , belongir stores , and thar stores . The Mrced cells , 138  that control the phosphorylation state of MLC2 enables lemented . On the nfection . This harophages , 143.C. trachomatis host cell infection of the 14-3-3 proteins and of its reported interactions with Inc proteins is unclear.The 14-3-3 phospho-serine/phospho-threonine binding proteins are present in all eukaryotic cells, where they have many and varied interacting partners and regulate a wide diversity of cellular processes . DiffereC. trachomatis-infected cells [Many Incs have relatively long stretches of their polypeptide chains predicted to be exposed on the host cell cytosol, while others have putative host cytosolic regions of less than 30 amino acid residues . This sued cells . Furthered cells  was alsoed cells .C. trachomatis incC mutant revealed an unstable vacuolar membrane [incV, ct224, and ct850 did not lead to unstable inclusions [Several of the Incs involved in heterophilic Inc:Inc interactions have been localized to ER-inclusion MCSs  or to inclusion microdomains . A membrane , consistclusions .in vitro serial passage of trachoma and genital C. trachomatis strains, but not of LGV strains, leads to the inactivation of the ct135 gene [. Because the ct135 gene is intact in low-passage genital clinical isolates, this indicates that, at least in these strains, ct135 is under positive selection in vitro, and negative selection in vivo [C. trachomatis serovar D strain with a single nucleotide insertion in the middle of the ct135 gene (which might correspond to a null mutation) is less virulent in a mouse infection model than an isogenic strain with a single nucleotide deletion in the beginning of the ct135 gene (which might enable the putative production of a near intact CT135 protein) [in vitro [C. trachomatis virulence in vivo.Different lines of evidence indicate that 135 gene –153, encprotein) , 150. Inin vitro . TherefoC. trachomatis proteins have been shown to be delivered outside of the inclusion and detected in the host cell plasma membrane, cytoplasm or nucleus, and/or at the inclusion membrane . Ten of these proteins have also been detected within the inclusion lumen, outside of the chlamydiae . Furthermore, although their delivery outside of the inclusion was never formally shown, there are C. trachomatis proteins  for which there is evidence for an effector role see  footnote. We will not consider C. trachomatis secreted proteins that likely form the T3S system translocon either in the host cell plasma membrane or in the inclusion membrane  [Besides Incs, 24 6/CopB2) , 155, or6/CopB2) , or that6/CopB2) , 157.C. trachomatis EBs contain functional T3S systems and are packed with effector proteins injected upon contact of the chlamydiae with host cells [.The invasion of host cells by EBs involves first interactions between different chlamydial adhesin molecules and diverse eukaryotic receptors . Furtherst cells , 158–161C. trachomatis induces protein tyrosine phosphorylation [C. trachomatis invasion, RAC1 recruits Wiskott-Aldrich syndrome protein family member 2 (WAVE2) and abl interactor 1 (ABI1), while CDC42 recruits Neural Wiskott-Aldrich syndrome protein (N-WASP), promoting the initiation of actin polymerization by the actin-related protein 2/3 (ARP2/3) complex [tour de force that led to the identification of TarP   was crucial for the ongoing clarification of how Chlamydia modulate all these processes [Invasion of host cells by rylation , 163, anrylation , 164. Thrylation , and on rylation . During  complex . The tourocesses .C. trachomatis revealed a tyrosine phosphorylated chlamydial protein [Yersinia as heterologous bacteria [C. trachomatis EBs use their T3S system to deliver an effector (TarP) into host cells that is then tyrosine phosphorylated, and which is associated with actin polymerization at chlamydial entry sites .Immunoprecipitation with an anti-phosphotyrosine antibody followed by mass spectrometry from protein extracts of cells infected by  protein . Becausebacteria . Furtherbacteria . This inC. trachomatis serovars and Chlamydia species [C. trachomatis TarP includes a tyrosine-rich repeat domain corresponding to the region that is tyrosine phosphorylated [C. trachomatis serovars and is absent in TarP from other Chlamydia species [C. trachomatis TarP is phosphorylated by Abl and Src family tyrosine kinases [.TarP is present in all  species , 169. C.orylated . The num species –170. Not kinases , 172, an kinases . In euka kinases . In the  kinases  , and a complex formed by ABI1, SOS Ras/Rac guanine nucleotide exchange factor 1 (SOS1), and epidermal growth factor receptor pathway substrate 8 (EPS8) [C. trachomatis entry into host cells, and are all required for chlamydial invasion [C. trachomatis invasion, the chlamydial uptake process is inhibited after expression in C. trachomatis of a TarP mutant protein lacking the tyrosine-rich repeats [C. trachomatis TarP binds and recruits RAC1 activators (VAV2 and the ABI1:SOS1:EPS8 complex), ultimately leading (through WASP2 and ABI1) to the initiation of localized actin polymerization by the ARP2/3 complex and promotion of chlamydial invasion of host cells [.The tyrosine-rich repeats of rization . Ectopicruitment . Further8 (EPS8) . The bin8 (EPS8) . The act8 (EPS8) . Accordi8 (EPS8) . VAV2, Sinvasion . In agre repeats . This do repeats , 178. Ovst cells   and vinculin during chlamydial invasion through protein motifs also present in TarP orthologues from other Chlamydia species, including C. trachomatis [C. pneumoniae TarP orthologue, which also binds vinculin [In summary, delivery of TarP into host cells by the T3S system in chomatis , 182. Fivinculin , can stavinculin .C. trachomatis TmeA (translocated membrane-associated effector A) and TmeB  as T3S effectors [The idea that chlamydial genes with mRNA levels first detected late in the chlamydial developmental cycle could be candidates for encoding proteins packed in EBs led to the identification of ffectors , 186.tmeAB operon [C. trachomatis EBs [Yersinia is T3S-dependent [C. trachomatis-infected cells revealed that TmeA and TmeB localize in the host cell cytoplasm, nearby the nascent inclusion, between 1-3 h post-infection [. The TEM-1 β-lactamase reporter assay, enabling to monitor the delivery of bacterial effector proteins into mammalian host cells [C. trachomatis-infected cells [C. trachomatis into host cells early in infection and they localize near the nascent inclusion. Later in infection, TmeA associates with the host cell plasma membrane and TmeB remains associated with the inclusion membrane.TmeA and TmeB are encoded by a small B operon . They aratis EBs , 186, anependent , 187. IFnfection , 186  .Many T3S effectors require specific bacterial cytosolic chaperones for their proper delivery into target host cells . TarP, Tproteins . This leYersinia in a T3S-dependent manner, which is promoted by Slc1 [C. trachomatis infected cells and localizes near the nascent inclusion [. Furthermore, as TarP, TepP is rapidly tyrosine phosphorylated by host cell Src family kinases after its chlamydiae-mediated delivery into infected cells [TepP is secreted by  by Slc1 , as also by Slc1 , 194. Asnclusion   genes [Two different cterized , 196. C.La cells , 196, buT) genes , 196.C. trachomatis-infected cells, TepP has been shown to bind the CRK proto-oncogene, adaptor protein (CRK), CRK like proto-oncogene, adaptor protein (CRKL), glycogen synthase kinase 3β (GSK3B), and different subunits of class I PI3K [. CRK, CRKL, and PI3K  are recruited to the proximity of the nascent inclusion in a TepP-dependent manner [IFIT genes in cells infected by TepP-expressing chlamydiae depends on PI3K [In s I PI3K , 196  in cultured cells, and the severity of the effect correlates with the multiplicity of infection [. trachomatis-infected cells [C. trachomatis up to 1 h post-infection [Chlamydial genomes encode proteins displaying homology with the large e toxins , which ie toxins . Some ofe toxins . This isnfection . Ectopiced cells . This efed cells . The CT1nfection . Furthernfection .C. trachomatis strains. Within the cytoplasm of infected cells, CT166 likely glucosylates and inactivates RAC1. This can potentially downmodulate the RAC1-mediated signalling resulting in actin cytoskeleton changes required for chlamydial invasion .In summary, while direct evidence is lacking, CT166 is probably delivered into host cells by EBs through the T3S system of some C. trachomatis CT622 protein was initially identified in the host cell cytosol and within the inclusion lumen [S. flexneri in a T3S-dependent manner [ct622 mutant strain (see below) [.The on lumen . The N-tt manner . Althougt manner , 201, itt manner , 201, anin vitro GGTase activity has not been detected for CT622 [C. trachomatis ct622 mutant revealed several defects during the chlamydial developmental cycle [C. trachomatis-dependent protein tyrosine phosphorylation is much reduced after infection by the ct622 mutant strain [C. trachomatis.The 3D structure of the C-terminal of CT622 has been determined by X-ray crystallography and revealed similarity with geranylgeranyl transferases (GGTases) and synthases . Proteinor CT622 . On the al cycle . The mutal cycle . Furthert strain . OverallC. trachomatis, a ubiquitin-based probe was used to search for possible deubiquitinases (DUBs) within infected cells [. Cdu1 or Cdu2 display DUB and deneddylase activities when ectopically expressed in mammalian cells [in vitro DUB and acetyltransferase activities of Cdu1 involve the same catalytic centre in the enzyme, and the dual specificity is conferred by a helix that can contact either ubiquitin or coenzyme A [The reversible post-translational modification of proteins by ubiquitination, or by other ubiquitin-like modifications like neddylation, is fundamental to control several eukaryotic cell processes and is often targeted by pathogens . In the ed cells . This eved cells , 205  [cdu1 mutant strain displayed a defect in a mouse model of infection [cdu2 null-mutant strain did not show a defect in the generation of infectious progeny in both A549 and HeLa cells [C. trachomatis-induced host cell phenotypes, the characteristic Golgi redistribution around the inclusion was not observed in cells infected by cdu1 or cdu2 null mutants [C. trachomatis InaC (see above), Cdu1 and Cdu2 are involved in Golgi redistribution during chlamydial infection .cterized , 206. Byll line) , but notnfection . In contC. trachomatis infected cells [. MCL1 is an anti-apoptotic protein [Chlamydia-infected cells to apoptosis [C. trachomatis-infected cells, MCL1 is stabilized by deubiquitination, preventing its degradation in the proteasome [. Moreover, in cells infected by wild-type C. trachomatis, deubiquitinated MCL1 accumulates around the inclusion, while in cells infected by the cdu1 mutant there is an increase in ubiquitinated MCL1 at the inclusion and the overall cellular levels of MCL1 are decreased [cdu1 mutant are not more sensitive to apoptosis than cells infected by the wild-type strain [cdu1-infected cells [The nuclear factor-κB (NF-κB) family of transcription factors controls several mammalian genes with important roles in immunity . Ectopiced cells   important for apoptosis-resistance in Chlamydia-infected cells.In summary, Chlamydiacea-specific domain of unknown function (DUF582) within their primary structure. In C. trachomatis, these DUF582-containing proteins are CT619, CT620, CT621, CT711, and CT712 . Although this has not been directly shown for all of them, it is generally assumed that the chlamydial DUF582-containing proteins are T3S substrates delivered into the cytoplasm of infected cells and that at least some of the DUF582 proteins are also transported into the host cell nucleus. This general concept derives from several experiments. First, DUF582-containing proteins from C. trachomatis, C. pneumoniae and C. caviae are T3S substrates, as deduced from experiments using S. flexneri as heterologous bacteria [C. trachomatis DUF582-containing proteins have been detected in the cytoplasm  and nucleus  of infected cells [. Finally, CT620, CT621, and CT711 also localize in the host cell nucleus after their ectopic expression in uninfected human cells [Each chlamydial genome encodes several proteins possessing a bacteria , 212. Seed cells   machinery of host cells [C. trachomatis DUF582 proteins and a component of ESCRT complexes (Hrs) was initially found in yeast two-hybrid screens using the C-terminal region of CT619 (containing the DUF582) as bait [C. trachomatis DUF582 proteins and Hrs, and between CT619 and TSG101 [C. trachomatis CT619, CT620, CT621, CT711, and CT712 can bind Hrs through their C-terminal DUF582 region, and CT619 can also bind TSG101 through its N-terminal region. The physiological significance of these interactions is presently unclear, as siRNA-mediated depletion of Hrs or TSG101 does not appear to interfere with C. trachomatis internalization or with the chlamydial developmental cycle [C. trachomatis proteins with components of the ESCRT machinery suggests that these chlamydial effectors could modulate the host cell endocytic pathway or other ESCRT-dependent processes. For example, the ESCRT machinery is also needed for host cell scission events, such as abscission during cytokinesis [C. trachomatis exit by extrusion [C. trachomatis DUF582-containing effectors regulate chlamydial extrusion.At a functional level, st cells , mostly st cells . The int as bait . Subsequ as bait . Furtherd TSG101 . Thus, Cal cycle . Neverthokinesis , and it xtrusion . It is, C. trachomatis, the presence of LDs around and within the inclusion [C. trachomatis-infected cells [Chlamydia. The relevance of LDs for chlamydial infections is further supported by their detection within inclusions from cells of mice that had been infected by the mouse and hamster pathogen C. muridarum [C. trachomatis growth in cells lacking LDs have produced conflicting data [Lipid droplets (LDs) are organelles playing an important role in lipid and energy homeostasis of eukaryotic cells . They arnclusion , 220, aned cells , 221, inuridarum . Howevering data , 224. Fuing data , 223, apC. trachomatis proteins have been shown to associate with these organelles. Phenotypic and subcellular localization screens of a large collection of yeast strains expressing C. trachomatis proteins revealed four chlamydial LD-associated (Lda) proteins [C. trachomatis-infected cells, Lda1, 2, and 3 co-localize with LD-like structures surrounding the inclusion [C. trachomatis-infected cells, providing some support to the model. Furthermore, ectopically expressed CT618 and Cap1 also associate with LDs in mammalian cells [Regardless of the role of LDs in chlamydial infections, several proteins , 219: Ldnclusion . Overallnclusion . Althougnclusion , 221, thC. trachomatis genome revealed a gene potentially encoding a protein containing a SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain [C. trachomatis SET domain-containing protein was deduced to be a T3S substrate based on secretion assays using S. flexneri as heterologous bacteria [C. trachomatis, the SET domain-containing chlamydial protein was found in the host cell nucleus associated with chromatin [in vitro system, NUE was capable of methylating histones H2B, H3 and H4 [C. trachomatis effector localizing in the nucleus of infected cells where it presumably methylates histones and therefore possibly remodels chromatin .The first ) domain , mostly ) domain . The C. bacteria . After bhromatin . The chlhromatin . Further3 and H4 . This shC. trachomatis T3S substrates using Yersinia as heterologous bacteria identified several candidate chlamydial effectors [C. trachomatis effector associated with the Golgi) [. However, as chlamydial infection of cultured cells advances in time, CteG is found progressively more associated with the host cell plasma membrane [C. trachomatis cteG insertional mutant was generated, but it did not show a chlamydial growth defect in cultured cells [cteG mutant did not display an alteration in Golgi redistribution around the inclusion [C. trachomatis effector with dual and sequential localization in infected cells, first associated with the Golgi and then with the host cell plasma membrane . CteG might interfere with host cell vesicular trafficking, but this remains to be shown in infected mammalian cells.A screen for ffectors . Additioffectors . The proe Golgi)   [C. trachomatis-infected cells [In vitro, transient concentration-dependent homodimerization of CPAF leads to autocleavage and formation of a stable homodimer [In addition to Cdu1 and Cdu2 (see above),  factor)  is proba factor) –237. Fol factor) , CPAF waed cells . Subsequed cells ). Howeveed cells . Nonetheed cells –244. In omodimer , 244. Thomodimer , 242.C. trachomatis CPAF has been detected in the cytosol of infected host cells by IF microscopy . H. HC. trapy , the CT847 effector (see above), and three other T3S system-related proteins [C. trachomatis [C. trachomatis of the p65 subunit of NF-κB [C. trachomatis is likely a consequence of the activity of CPAF on other effectors or T3S system-related proteins [A  progeny , 248, inchomatis , 248, anchomatis . It is tC. trachomatis OmcB [cpaf null mutant strains, CPAF has been also associated with inhibition of host cell cytokinesis [C. trachomatis lytic exit [Other possible targets of CPAF in the inclusion lumen are tis OmcB , which ctis OmcB , and PI1tis OmcB , in the tis OmcB . In the tis OmcB . Based ookinesis  and withtic exit . Althougtic exit , these Ctic exit , 251.C. trachomatis survival in the mouse lower genital tract [C. trachomatis cpaf mutant shows defects in mice infection models, these defects are suppressed in neutrophil-depleted or in FPR2-knock-out mice [During chlamydial lytic exit, the cytoplasmic and inclusion contents, including CPAF, are also released. Different data support that extracellular CPAF promotes evasion of the host innate immune response –254, whial tract . CPAF clal tract . Accordiout mice . Furtherout mice , and comC. trachomatis secretes a potent serine protease (CPAF) with functions within host cells, during the chlamydial developmental cycle, as well as in the extracellular space, after the release of the chlamydiae from infected cells. CPAF is conserved in Chlamydiaceae, and in Chlamydia-like organisms infecting amoeba there are homologues showing ~30% of amino acid sequence identity to C. trachomatis CPAF [Chlamydiales.In summary, tis CPAF . TherefoC. trachomatis proteases that have been implicated in the subversion of host cell processes.Chlamydial high temperature requirement protein A (HtrA)  and tailC. trachomatis HtrA, in addition to its expected functions in chlamydial physiology and development [C. trachomatis HtrA possesses a signal peptide recognized by the Sec system [C. trachomatis HtrA in the host cell cytosol, there is no additional evidence for a possible function of the protein in subverting host cell functions.HtrA serine proteases are conserved in unicellular and multicellular organisms where they mostly play an important role in protein quality control . In Gramelopment , the proelopment . C. tracc system . Given tC. trachomatis, Tsp has a chaperone activity and functions in protein quality control [C. trachomatis Tsp has never been detected in the host cell cytosol, ectopically expressed Tsp can cleave the p65 subunit of NF-κB [in vitro pull-down assays [C. trachomatis-infected cells under controlled conditions of cell lysis [Tsps are present in many bacteria where they have diverse housekeeping roles and act by processing the C-terminal region of different target proteins . In C. t control . Althougof NF-κB , and Tspn assays . Howeverll lysis , and thell lysis .Chlamydia species possess a cryptic plasmid encoding the so-called plasmid glycoproteins (pGPs 1-8) and two small RNAs [C. trachomatis pGP3 has been found associated with the outer membrane of EBs [. pGP3 is also found in the cytosol of cells infected by other Chlamydia species carrying the virulence plasmid [Most all RNAs . C. trace of EBs , and alse of EBs  . Transport of CT311 and CT795 from the chlamydiae into the host cell cytosol can be prevented by a small molecule inhibitor of the Sec system [, either in C. trachomatis-infected cells or after its ectopic expression [The ns CT311  and CT79ns CT311  were alsc system , 278. Asc system , 278, thpression .C. trachomatis proteins that have been detected in the inclusion membrane but do not possess a bilobed hydrophobic region, Cap1 and CopN are normally classified as Incs [Among the  as Incs , 59.C. trachomatis-specific CD8+ T-cell line [Cap1 was identified as a protein accessing the host cell cytosol based on a screen for chlamydial proteins recognized by a ell line . Cap1 waell line , which well line , 63. Litell line .C. trachomatis T3S system gatekeeper, and it was the first chlamydial protein shown to be a T3S substrate [C. trachomatis CopN could be strictly associated with its regulatory function, but a possible effector role should not be disregarded. For example, C. pneumoniae CopN has been shown to interfere with host cell microtubules [C. trachomatis CopN does not display this activity on microtubules [Several bacteria harbouring T3S systems have a so-called gatekeeper protein required for secretion of the T3S system translocators and controlling the beginning of effector delivery into host cells, which can involve secretion of the gatekeeper itself . CopN isubstrate . It was ubstrate , which wubstrate . This lootubules , 283; hootubules .C. trachomatis proteins that localize in the host cell cytoplasm or nucleus, or in the inclusion membrane, and that are also found in the lumen of the inclusion  and hydrolysis  [.nd MalQ) . For glyChlamydia virulence plasmid-dependent accumulation of glycogen in the lumen of the inclusion of C. trachomatis and C. muridarum [glgA [S. flexneri [C. trachomatis strains with mutations in GlgB show an accumulation of aggregates within the inclusion possibly corresponding to unbranched glycogen [. Differently from other bacterial glycogen synthases, chlamydial GlgA can use both ADP- and UDP-glucose as substrate [C. trachomatis-infected cells, the host transporter SLC35D2 is recruited to the inclusion membrane and likely mediates the transport of UDP-glucose into the lumen of inclusion [. Thus, C. trachomatis should mediate the T3S-dependent transport of GlgA and GlgB into the inclusion lumen, and the ability of GlgA to use UDP-glucose as substrate enables the chlamydiae to synthesize glycogen in this confined compartment . However, the C. trachomatis T2S system should also play a role in the chlamydial glycogen metabolism because a strain with a mutation in an essential component of this protein transport system shows abnormal glycogen aggregates within the inclusion [C. trachomatis inclusion also depends, but to a lesser extent, on its transport in bulk from the host cell cytosol , which brings along the host glycogen synthase Gys1 [There is a uridarum , 285. Thum [glgA , 287. Fuum [glgA , and theum [glgA . In addiflexneri , and C. glycogen . Altogetubstrate . Accordinclusion  , which also possesses a T3S signal recognized by S. flexneri [.It is unclear how the accumulation of glycogen within the inclusion benefits chlamydiae. By one side, a plasmidless  progeny , 286. On progeny . C. traccroscopy . Therefohosphate , its conflexneri    proteins [. Pls1 and Pls2 lack obvious Sec-dependent signal peptides [C. trachomatis-infected cells with anti-Pls1 and anti-Pls2 antibodies inhibits inclusion expansion, suggesting that Pls1 and Pls2 could be important for C. trachomatis and/or inclusion growth [proteins . By IF mproteins  , are amongst the C. trachomatis chromosomal genes more clearly upregulated by the Chlamydia virulence plasmid through pGP4 [In summary, Pls1, Pls2, CT142, CT143, and CT144 have unknown function but they are secreted into the lumen of the inclusion where they appear as globular structures, as detected by IF microscopy ugh pGP4 , 295.C. trachomatis has been shown to deliver at least ~60 proteins into the inclusion membrane and cytoplasm of host cells that function as effectors (Tables 1 and 2). Moreover, there are numerous putative Incs that have never been localized to the inclusion membrane [C. trachomatis proteins delivered into the inclusion membrane and host cell cytoplasm might be ~ 70-90. In addition, C. trachomatis also secretes proteins into the inclusion lumen . Considering that a typical C. trachomatis genome encodes ~ 900 proteins [membrane , 43, andmembrane , 227. Evproteins , > 7%, aC. trachomatis genomes are relatively small (~ 1 Mb) and underwent extensive reductive evolution [Legionella pneumophila [C. trachomatis effector gene has been shown to be essential, and the analysis of some effector gene mutants suggests possible redundancy. Overall, this indicates that some type of redundancy might also be relevant among chlamydial effectors.A feature of pathogenic bacteria delivering high numbers of effectors into host cells is redundancy, which can be, for example, effectors with similar activity and functions or effectors with different activity but targeting the same host cell process . Considevolution , 298, itumophila . HoweverChlamydia, horizontal gene transfer is less important, but not irrelevant [C. trachomatis. However, genes encoding Inc proteins and other T3S substrates have been shown to be encoded within operons [Another feature of pathogenic bacteria carrying several effector genes is that some of them were acquired through horizontal gene transfer –301. Givrelevant , in chla operons , 294. ApC. trachomatis delivers at least six effector proteins  into the cytoplasm of host cells from extracellular adhering EBs and/or shortly after their internalization . Some of these effectors are important for chlamydial invasion , host cell survival (TarP) and immune signalling (TepP). Furthermore, TarP [Salmonella effector proteins [C. trachomatis effectors packed in EBs downmodulate the alterations in the actin cytoskeleton that promote chlamydial invasion. An obvious candidate is CT166, which can potentially inactivate the TarP-dependent activation of RAC1. However, while the activity of Salmonella effectors causes reversible changes in Rho family proteins [C. trachomatis strains. Another key aspect that remains unclear is how the nascent inclusion limits interactions with the endosomal pathway to prevent chlamydial destruction in a phagolysosome. This early subversion of the endocytic pathway occurs even in the absence of chlamydial protein synthesis [re, TarP , 186, CTre, TarP , 201, Tmre, TarP , and Tmeproteins , some ofproteins , the gluynthesis . Therefoynthesis .C. trachomatis uptake by host cells, chlamydial gene expression leads to the production and secretion of effector proteins that influence the subsequent stages of the developmental cycle. This group of effectors is largely comprised by early-cycle Incs, which modulate interactions with host cell vesicular trafficking (IncE and CpoS), help the migration of the nascent inclusion to the centrosomal region (CT850), establish ER-inclusion MCSs and promote the non-vesicular transport of sphingomyelin into the inclusion (IncV and IncD), and mediate inclusion membrane stability . IncV might even be delivered earlier into the inclusion membrane as it is present in EBs [C. trachomatis modulates these processes remains to be established. For example, it is still unclear how the inclusion intercepts vesicular trafficking from the TGN to selectively acquire sphingolipids and cholesterol [After t in EBs . A detailesterol –117, 305lesterol , 306.Chlamydia virulence plasmid, likely through the regulation of expression of T3S effectors and/or of CPAF [C. trachomatis effectors concomitantly released from the inclusion lumen and/or host cell cytosol continue to function extracellularly, as is the case of evasion of the innate immune response by CPAF, and possibly by pGP3. Evidently, much remains to be understood about the subversion of all these processes by C. trachomatis.As the developmental cycle continues, several other chlamydial effectors are delivered into the cytoplasm of host cells. These effectors mediate fusion between inclusions (IncA), continue subverting host cell vesicular trafficking , modulate microtubules (IPAM) and mediate their modification (InaC), promote the assembly of F-actin (InaC) and the redistribution of the Golgi around the inclusion , and possibly promote the acquisition of LDs by the inclusion , interfere with host cell transcription (NUE), and modulate host cell death (CpoS and Cdu1). Finally, chlamydial host cell exit is also controlled by Incs (MrcA and CT228), and by the  of CPAF , 295. EvC. trachomatis-infected host cells. For this, C. trachomatis secretes proteins into the inclusion lumen synthesizing and hydrolysing glycogen [en route to the inclusion membrane or host cell cytoplasm. Furthermore, even if chemical fixation in preparation of samples for microscopy can create artifacts [C. trachomatis T2S system. Assuming this is the case, it is unknown how these proteins can reach the host cell cytoplasm before the permeabilization of the inclusion membrane that precedes chlamydial lytic exit.The inclusion lumen is a functionally and metabolically important compartment of glycogen , mediatiglycogen , with unrtifacts , host certifacts , differertifacts , 309, anrtifacts  and perortifacts  have beertifacts , 278. Hortifacts  and deduC. trachomatis into the inclusion membrane and host cell cytoplasm are transported by the T3S system. C. trachomatis EBs appear to be better equipped for T3S than RBs [. Surprisingly, some C. trachomatis T3S substrates are transported into the inclusion lumen [C. trachomatis mutant with an essential T3S system gene inactivated would be non-viable. Therefore, although a formality in most cases, final demonstration that Incs and other chlamydial proteins are T3S substrates will require the generation of a chlamydial conditional mutant, which has not been described yet.Most, if not all, of the proteins delivered by than RBs , suggestthan RBs  (Figure on lumen , 294. Coon lumen . FinallyC. trachomatis delivers many effector proteins into host cells, and into the lumen of the inclusion, and these proteins interfere with a wide diversity of host cell processes to promote chlamydial invasion, survival, growth, development and dissemination. Although enormous progress has been achieved in recent years elucidating the functions of these chlamydial secreted proteins, we are most likely only seeing the tip of the iceberg. The generation of C. trachomatis mutants on relevant genes and their careful characterization in infected cells and in animal models of infection, together with molecular, cell and structural biology studies, will certainly provide explanations to some of the current outstanding issues and originate new concepts as well as additional questions. It might also establish a relation between effector function and C. trachomatis-related pathogenicity. This will expand our fundamental understanding of host-pathogen interactions and of chlamydial and host cell biology, which is critical to devise novel prophylactic and therapeutic approaches against infectious diseases.


Paratylenchus lepidus and P. minor were detected in the rhizosphere of Elaeocarpus sp. and Chinese red pine from Taizhou and Hangzhou, Zhejiang Province, China. Previously, P. lepidus has been reported from China whereas P. minor was originally described from India decades ago in the rhizosphere of peach but was never reported thereafter. In this study, both species were characterized morphologically and molecularly coupled with SEM observations. Morphologically, both species have four incisures in the lateral field, vulval present (SEM observations), stylet less than 30 μm long and cephalic region without submedian lobes. Phylogenetically, both species grouped with paratylenchid species having short stylets. Both species can be differentiated from each other by the shape of lip region (rounded in P. lepidus and narrow truncated in P. minor) and tail terminus (pointed in P. lepidus and a broadly rounded in P. minor) and several morphomemtrical values.The study provided an updated description of P. lepidus and P. minor and a first record of P. minor from China. In addition, both species are the first paratylenchid species recorded from Elaeocarpus sp. and Pinus tabuliformis, respectively.Two populations of  Gracilacus and Paratylenchus based on stylet length (Paratylenchus species were detected in a soil sample of Elaeocarpus sp. and Chinese red pine (Pinus tabuliformis Carr). The preliminary studies revealed the status of these species as P. lepidusP. minor  to provide the updated description with morphological and molecular characterization, (ii) to elucidate important morphological details through SEM observations, (iii) to investigate the phylogenetic position and to locate the closely related Paratylenchus species.The species of pin nematodes are commonly distributed in China, more than 30 species have been reported from 21 provinces . The host length . The spet length . The mort length . Howevert length . For pret length , 18S  method was used within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees  using 50% majority rule.The sequences were deposited into the GenBank database. DNA sequences were aligned by MEGA7  using default settings. The DNA sequences were compared with those of the other pin nematode species available at the GenBank sequence database using the BLAST homology search program. The model of base substitution was evaluated using MODELTEST . The Akaike-supported model, the base frequencies, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0  running the chain for 1 × 10SystematicsFigs. –2P. lepidus Taizhou population are given in Measurements of the females of ca 70% of the total stylet length; stylet knobs rounded, posteriorly directed; dorsal oesophageal gland opening 5 to 6 μm behind stylet base; median pharyngeal bulb elongate, bearing distinct large valve; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, cardia inconspicuous; excretory pore position slightly anterior to or at midway of basal pharyngeal bulb; hemizonid position located immediately anterior to the excretory pore; gonad short, prodelphic, spermatheca rounded; vulva a transverse slit occupying half of the body width, vulval lips not protruding with prominent advulval flap; post uterine sac absent; anus indistinct; tail slender, finely annulated, relatively curved, gradually tapers to form a finely rounded to bluntly pointed terminus.Body slender, not obese, ventrally arcuate when heat relaxed; cuticle finely annulated; lateral field with four incisures; SEM observations showing a somewhat rectangular labial disc with two distinct amphid openings, submedian lobes absent and oral aperture slit like surrounded by semi-globular shaped projections positioned on the labial plate; cephalic region narrow, flattened rounded, not offset from body; cephalic sclerotization weak; stylet short, cone Not found.Elaeocarpus sp. from Taizhou City, Zhejiang Province, China. The geographical position of the sampling site is 120°17'23 E; 29°4'30 N.This population was found in the rhizosphere of P. lepidus was originally described from Sri Lanka associated with the tea plants  is in a clade with an unidentified Gracilacus (MF095023) species from the USA and P. straelini (AY284631). This clade is sister to a clade comprised of P. conicephalus (KP966493), P. dianthus (AJ966496), P. goldeni (KJ934186), P. microdorus (AY284633), P. nanus (KJ636435), P. similis (KJ636432), P. projectus , and P. cf. neoamlicephalus (AY284634). All of these species have 4 lateral lines, advulval flap and stylet less than 40 µm except P. straelini has stylet longer than 40 µm. P. minor (MK660189) is in a second clade and is grouped with P. shenzhenensis  and it further grouped with species having longer stylet i.e. Gracilacus colinus , G. paralatescence , and G. wuae . Apparently, both species grouped with species having shorter stylets and the presence of advulval flap.Based on the phylogenetic analysis of the 18S gene Fig. , the pinP. lepidus (MK886692) is identical with an unidentified Paratylenchus sp. (MH156807) from Fujian Province and it is grouped with unidentified Paratylenchus sp. from Zhejiang (JX109836) and Paratylenchus sp. from USA (KF242230-32). This clade is sister to a clade comprised of P. dianthus (KF242229), P. straeleni (KM875547), P. hamatus (KF242218), P. tenuicaudatus (KU291239), and P. nanus , all of these species have short stylet, advulval flap and four lateral lines except P. straeleni this species also have advulval flap and four lateral lines but stylet is 44 to 66 μm long. The pairwise similarity between P. lepidus and unidentified Paratylenchus sp. (MH156807) from Fujian, Paratylenchus sp. (JX109836) from Zhejiang and Paratylenchus sp. from USA (KF242230-32) are 99, 91 and 92% with 2, 59 and 64 bp difference.Based on 28S gene Fig. , P. lepiP. minor (MK660188) is nearly identical with an unidentified Paratylenchus species (KT258978) from Guangdong Province. This clade is grouped with P. aquaticus (KF2422439-41), P. leptos (KR270602) and P. rostrocaudatus (KR270601). These species have short stylet and have advulval flap, but the number of lateral lines is not the same, i.e. 3 for P. leptos and P. rostrocaudatus, and 2 for P. aquaticus. This clade is further grouped with species having longer stylets and absence of advulval flaps but the number of lateral lines is not consistent, i.e. 3 for P. aculentus and G. paralatescence and 4 for the G. wuae. The pairwise similarity between P. minor and unidentified Paratylenchus sp. (KT258978) from Guangdong is 99% with 9 bp difference.P. lepidus (MK886695) is identical with P. lepidus (EF126178) from Taiwan and further grouped with P. lepidus (JX992859-60) from Hunan and an unidentified Paratylenchus sp. (KF242273-74) from USA, but with significant difference based on branch length. P. minutus (EF126180) is in the same clade as P. lepidus from Hunan. P. dianthus  is sister to this clade. This clade is further grouped with the unidentified Paratylenchus (KF242243) species from the USA, P. hamatus (KF242257), P. nanus , P. labiosus (JQ708154) and P. goldeni (KJ934186). All of these species are reported to have shorter stylet, advulal present and four lateral lines. The ITS tree revealed that the P. lepidus from Hunan might be a different species, as indicated by the ITS pairwise sequence identity between P. lepidus from Zhejiang and Hunan population, i.e. only 83% identical with 156 bp difference. Considering this we propose that P. lepidus was only reported from Taiwan and Zhejiang. The two unidentified Paratylenchus species (KF242273-74) from the USA possibly be similar to that of P. lepidus from Hunan. The pairwise sequence similarities of P. lepidus from Hunan and Paratylenchus species (KF242273-74) from USA is 99% with 2 bp difference.Based on ITS gene Fig. , our popP. minor is nearly identical with an unidentified Paratylenchus species (KT258979) from Guangdong Province and it is sister to P. leptos (KR270605) and P. rostrocaudatus (KR270604), both of these species have short stylets and advulval flap but the number of lateral lines is three. This clade is further grouped with the species having longer stylets, i.e. G. paralatecence (MH200615), P. aculentus , P. nanjingensis (KM 366103), G. wuae (KM061783), P. guangzhouensis (KT725653), G. bilineata (EU247525), and P. aquaticus (KF242278). The pairwise sequence identities between P. minor and unidentified Paratylenchus species (KT258979) from Guangdong Province is 98% with 10 bp difference.P. lepidus and P. minor are in two well-separated clades and grouped with species having shorter stylets and advuval flap, the species having longer stylets arranged at the basal position of the trees. To this point we suppose only fewer paratylenchid sequences were available for the phylogenetic studies, the inclusion of more sequences from other known species will be helpful to provide a better resolution of paratylenchid phylogeny.Our phylogenetic tree based on 18S, 28S and ITS tree gene sequences revealed that the P. alleni (P. bukowinensis (P. epacris (P. minutus (P. nanjingensis (G. steineri (Pinus massoniana, P. sylvestris and P. thunbergii (P. minor is the first paratylenchid species found in the rhizosphere of Pinus tabuliformis which is a native pine species of northern and southern China (Paratylenchus species was ever found from the Elaeocarpus sp. in China. The discovery of these two pin nematodes expanded the distribution and host range. It is also noted that the recent discovery of several new species of paratylenchids from China suggests that presumably only a small fraction of the actually existing taxa is known so far. There are still large geographical areas insufficiently studied for their diversity.The host range of pin nematodes in China is quite diverse. The paratylenchid species have been documented from plants, shrubs and trees . Out of . alleni ; P. bukowinensis ; P. epac minutus ; P. nanjingensis , and G. steineri  were docunbergii . In thisrn China . None of


It has long been recognized that the process of preserving biological specimens results in alterations of body shape, though detailed studies examining the degree to which morphological changes occur throughout the preservation process are lacking. We utilize geometric morphometric analyses, an increasingly common tool for examining shape variation in a wide variety of biological disciplines, to examine the effects of formalin and ethanol preservation on the body shape of 10 freshwater fish species over time: from fresh specimens to eight weeks after preservation. We found significant changes in body shape among fresh and formalin fixed specimens. Furthermore, changes in body shape continue to occur after subsequent ethanol preservation. Two fish species collected at multiple localities show significant morphological differences for a limited number of morphometric characters. However, the significance, or lack thereof, often changed inconsistently from one stage of preservation to another. We conclude that morphometric analyses would ideally be performed on fresh specimens. However, recognizing that this is not always feasible, it is important to be aware of the morphometric changes that can occur during preservation. Alterations in body shape due to preservation  are manifested in a variety of ways . PreviouDespite the wide usage of preserved specimens, the effects of preservation are often not discussed as a limitation or potential source of error in studies that assess body shape e.g., ,21–23). –23. 21–2The results of previous studies ,20 were The purpose of the current study was to document the degree to which changes in body shape due to preservation occur over time in disparately related fish species, and to determine whether morphometric analyses performed at various stages of preservation time might influence any inferences drawn from such analyses. To accomplish this, we used geometric morphometric analyses to address two objectives. Our first objective was to assess changes in body shape among and between 10 species of fishes representing three different families and six genera throughout the preservation process; preserved specimens were examined in two-week increments over eight weeks. Our second objective was to quantify morphological changes in two fish species from different collection locales over an eight-week preservation process.Cyprinella lutrensis, C. venusta, Macrhybopsis hyostoma, M. marconis, Notropis chalybaeus, N. amabilis, Gambusia geiseri, Etheostoma spectabile, http://txstate.fishesoftexas.org/etheostoma%20fonticola.htmPercina apristis, and P. carbonaria. Sample sizes ranged from six to 74 individuals per species (All samples were collected under a Texas Parks and Wildlife Scientific Permit (SPR-0601-159), and study protocols and methods were approved by Texas State University Institutional Animal Care and Committee (1207-0109-01). A total of 10 fish species from disparately-related taxonomic groups were sampled for geometric morphometric assessment:  species . Individgeomorph [Thin-plate spline (TPS) files of the images were created with tpsUtil . Digitageomorph .procD.allometry function; 10,000 iterations) and removed from the data by obtaining size-adjusted residuals [GeometricMorphometricsMix for each species [C. venusta, C. lutrensis, and M. marconis, or at least the number of axes which avoided the issue of a singular matrix, for the remaining species, were used in this analysis. With this test we tested for differences between field and 2W, field and 8W, and 2W and 8W; these were chosen to see if there are significant differences in morphology after formalin fixation, overall preservation changes, and after ethanol preservation, respectively. A classical Bonferroni correction was applied for each species. Shape changes associated with PC axes 1 to 5 were visualized and plotted for each species representing individuals that had the minimum and maximum PC value. Shape change plots (mean shape for each time period) were calculated and constructed from the size-adjusted residuals for each time period using the function shape.predictor [A generalized Procrustes analysis (GPA) was performed first for each species separately to compute Procrustes coordinates for analysis, and subsequently checked for outliers ,33. Landesiduals . The siz species ,36,37. Aredictor . Each tigeomorph package. A repeated measures generalized linear model (GLM), using the function “lme”, was performed in R with the nlme package to obtain estimates of centroid size by treatment and determine whether centroid sizes change over time [Centroid sizes, the measurement of overall body size, were calculated as the square root of the sum-squared distances from landmarks to the centroid  and werever time ,40.C. lutrensis and C. venusta collected from multiple sampling locations were used to quantify morphological differences among sites and across preservation time periods. Both species were chosen because they had sample sizes greater than five individuals for each location  was performed on the first two PC axes to statistically compare  PCA scores of these axes between the different time periods using the vegan package in R [Samples of location . Separatage in R .M. hyostoma  was associated with PC1 and the abdominal region (being enlarged or shrunken) was associated with PC1 through PC5. The caudal region (changes in length or width) was associated with PC1 through PC3, head (length or tilt) was associated with PC4 and PC5, and lastly the eye (size) was associated with PC4 . RepeateP. carbonaria, indicating body changes over preservation time relative to field specimens  among collection locales for examined . For the periods .C. lutrensis, and the lowest Procrustes correlations were between field and 2W, 4W, 6W, 8W  may indicate the formalin stage of preservation tends to expand muscular body areas such as the caudal region and pectoral girdle, yet after subsequent ethanol preservation, these areas may become dehydrated and decrease in size , althougP. carbonaria) could have an impact on our analyses, however, the results are mostly consistent with other species which had larger sample sizes.There are potential sources of error in this study, which apply to geometric morphometric studies in general. Some of the trends seen in changes in body shape could be due to a jar effect, where storage influenced specimens’ body shape, potentially causing them to arch . HoweverC. lutrensis and C. venusta occurred throughout the preservation time periods based on their collection sites over the eight weeks, which could influence conclusions derived from these data. If the field time period was examined, one would have come to different conclusions than if one were to have used photographs from any of the preserved time periods. For example, at the field time period of C. lutrensis, one could have concluded that Cuero and Gonzalez possess significantly different body shapes. In contrast, if one used the specimens after two weeks of preservation, we would have concluded they were not significantly different morphologically. Additionally, the magnitude of changes differed between pairwise site comparisons and time periods relative to field. The largest absolute percent change in Procrustes distances occurred at 6W relative to field and the wide standard deviations indicate a high amount of variability in body shape. These changes in body shape were due to the preservation process because the same individuals from the same populations were used and compared over the different time periods.Many studies have performed geometric morphometric analyses using preserved fishes in a variety of biological, ecological, or evolutionary contexts ,14,21–23Overall this study demonstrates fish body shapes vary within the same species over preservation time, across different species, and within the same species by collection locale. Future studies comparing differences in body shapes should be aware of the potential effects that preservation has on morphology across different taxonomic levels and collection locales and take caution when performing morphometrics comparing fresh and/or preserved specimens. If possible, we would recommend the use of fresh specimens. Since this is not always possible, depending on the taxa being studied, setting aside a subset of specimens and assessing changes after preservation relative to fresh field could be performed  or if boS1 TableC. lutrensis and C. venusta for each sampling site used in pairwise site comparisons.Sample sizes for (DOCX)Click here for additional data file.S2 TableResults from the generalized linear models testing centroid size estimates by time periods for all cyprinids and poeciliids. Included are the slope estimate, standard error, degrees of freedom, t-value, p-value, and centroid size estimate from the model. Field represents measurements taken on freshly dead specimens, followed by preservation of two weeks (2W) through eight weeks (8W).(DOCX)Click here for additional data file.S3 TableResults from the generalized linear models testing centroid size estimates by time periods for all percids. Included are the slope estimate, standard error, degrees of freedom, t-value, p-value, and centroid size estimate from the model. Field represents measurements taken on freshly dead specimens, followed by preservation of two weeks (2W) through eight weeks (8W).(DOCX)Click here for additional data file.S4 TableC. lutrensis within each time period.Procrustes distances calculated for each pairwise site comparison for (DOCX)Click here for additional data file.S5 TableC. venusta within each time period.Procrustes distances calculated for each pairwise site comparison for (DOCX)Click here for additional data file.S6 TableC. lutrensis between field and all subsequent time periods. Included are pairwise percent changes for each pairwise site comparison, the mean, standard deviation, absolute mean, and absolute standard deviation (STDEV) for each time period comparison.Absolute percent changes in procrustes distances for each pairwise comparison for (DOCX)Click here for additional data file.S7 TableC. venusta between field and all subsequent time periods. Included are pairwise percent changes for each pairwise site comparison, the mean, standard deviation, absolute mean, and absolute standard deviation (STDEV) for each time period comparison.Absolute percent changes in procrustes distances for each pairwise comparison for (DOCX)Click here for additional data file.S1 FigC. venusta, B) G. geiseri, C) C. lutrensis, D) M. marconis, E) N. amabilis, F) N. chalybaeus, G) P. apristis, H) P. carbonaria, I) E. spectabile. Error bars represent one standard deviation.Mean of each principle components axis 1 and 2 for each species: A) (ZIP)Click here for additional data file.S2 FigC. venusta, B) G. geiseri, C) M. hyostoma, D) C. lutrensis, E) M. marconis, F) N. amabilis, G) N. chalybaeus, H) P. apristis, I) P. carbonaria, J) E. spectabile.Shape plots of minimum (black lines and points) and maximum (dark gray lines and points) PC axis values for PC 1–5 for each species. A) (ZIP)Click here for additional data file.S3 FigC. venusta, B) G. geiseri, C) C. lutrensis, D) M. marconis, E) N. amabilis, F) N. chalybaeus, G) P. apristis, H) P. carbonaria, I) E. spectabile.Trajectory analysis of time period relative to field for each species; shows mean shape at a particular time period  of preservation. A) (ZIP)Click here for additional data file.S1 DatasetC. lutrensis and C. venusta are river and site of capture.Included additionally for (ZIP)Click here for additional data file.


Cryptococcus gattii represents an emerging fungal pathogen of immunocompromised and immunocompetent hosts in the United States. To our knowledge, this is the first case of posttransplant immune reconstitution syndrome due to C. gattii meningoencephalitis successfully treated with corticosteroids. We also report successful maintenance phase treatment with isavuconazole, a novel triazole, following fluconazole-induced prolonged QT syndrome. A 55-year-old male with history of renal transplantation 9 years earlier presented to the hospital with confusion, headaches, neck pain, cough, nausea, and vomiting. The patient, now disabled, previously was an airline mechanic who had travelled remotely to China, the Philippines, Hong Kong, and Surinam. No significant changes to his immunosuppressive regimen were noted in the months prior to admission .6/mL, creatinine of 1.3 mg/dL (baseline creatinine 1.2 mg/dL), and non-reactive fourth generation HIV antigen/antibody test. A lumbar puncture performed upon admission showed a cerebrospinal fluid (CSF) opening pressure (OP) of 36 cm H2O , WBC of 70 cells/μL (62% lymphocytes), glucose 25 mg/dL, and protein 115 mg/dL; CSF India ink stain was positive for yeast forms, meningoencephalitis CSF polymerase chain reaction  was positive for Cryptococcus neoformans/gattii, and culture yielded Cryptococcus gattii. A cryptococcal antigen was reactive at a titer of 1:2560 in serum and CSF. Dilated fundoscopic examination was negative for signs of cryptococcal intraocular invasion. Computed tomography (CT) scan of the chest demonstrated 2 solid nodules in the right lung (Cryptococcus (Admission laboratory tests showed a white blood cell count (WBC) of 7.2 x 10ght lung . Biopsy tococcus .He received liposomal amphotericin B and flucytosine as induction therapy. Tacrolimus was restarted after day 5 of his hospitalization . Given p2O in his CSF , and an OP of 15 cm H his CSF . Althougf values . Dexametf values . TransitC. gattii represents an emerging fungal pathogen that inhabits eucalyptus-type flora, soil, and bird droppings in tropical areas. A North American outbreak of C. gattii was observed in the Pacific Northwest in 2004 [C. gattii infection (versus C. neoformans) was distinguished by a higher MIC to fluconazole and its ability to promote aggressive CNS infection [C. gattii infection in immunocompetent hosts [C. gattii given the low fluconazole MIC and absence of travel to the Pacific Northwest. He likely acquired C. gattii either domestically or during international travel earlier in his life. Prior global surveys show VGI and VGIII subtypes predominate in Asia and North America (except the Pacific Northwest), respectively [ in 2004 . C. gattnfection . It has nt hosts . Multilont hosts . Althougectively .C. gattii infection caused his disseminated infection. C. gattii infection in solid organ transplant (SOT) recipients shows smaller nodular or interstitial pulmonary infiltrates, more frequent leptomeningeal enhancement, and higher rates of intensive care unit admission and mortality (70%) [We suspect reactivation of latent ty (70%) . Conversty (70%) , a resulty (70%) .C. neoformans and by limiting the interaction of calcineurin binding protein 1 (CBP1) and calcineurin [C. gattii infection include female sex, CNS disease, and higher median CD4 counts [C. gattii-induced posttransplant IRS.Immune reconstitution syndrome represents a paradoxical worsening of signs and symptoms after an initial response (despite effective therapy and microbiologic cure) and cannot be explained by a persistent primary infection, new secondary infection, or adverse drug events. Posttransplant IRS following cryptococcosis is seen in 5%–11% of patients approximately 4 weeks to 9 months after initiation of antifungal therapy . Manifescineurin , 18. Ris4 counts . In our C. gattii infection are currently limited to case series and reports [The immunologic mechanisms mediating posttransplant IRS are obscure. It is suggested that the reduction of immunosuppression and administration of antifungal therapy favor a shift from an anti-inflammatory Th2/Treg-mediated cytokine response to a proinflammatory Th1/Th17-mediated response. It is this overzealous reversion to a proinflammatory response that potentially promotes IRS in the wake of cryptococcal clearance. Prevalence estimates of IRS during  reports . Interes reports . WhetherC. neoformans meningoencephalitis [In patients diagnosed with IRS, corticosteroids are employed to tip the net state of immunosuppression towards an anti-inflammatory response. Precedence for the treatment of posttransplant IRS in cryptococcosis with corticosteroids is limited to case reports . Aside fphalitis . Corticophalitis . Infectiphalitis . In our C. gattii isolate was not obtained; although, to date, our patient has experienced a successful clinical response to this agent. The therapeutic efficacy of isavuconazole in treating CNS cryptococcosis is limited. The VITAL trial showed a successful response to isavuconazole in 6 out of 9 patients treated for cryptococcosis [C. gattii associated CNS infection (none with SOT), and all with successful responses. Prior in vitro susceptibility data indicates low epidemiological cutoff values to isavuconazole and effective brain tissue penetration of radiolabeled isavuconazole in healthy wistar rats [C. neoformans meningoencephalitis [C. gattii meningoencephalitis in humans is unknown.His course was complicated further by the development of prolonged increase in the QTc interval, which correlated with the start of fluconazole  and 1E. coccosis . Three otar rats , 32. Isaphalitis . WhetherOpen Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ofz460_Suppl_Supplementary_FiguresClick here for additional data file.