Escherichia coli, while another bacterium, Proteus mirabilis, is more often associated with complicated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent requirements for specific central pathways in vivo despite colonizing and occupying the same host environment. Using mutants of specific central metabolism enzymes, we determined glycolysis mutants lacking pgi, tpiA, pfkA, or pykA all have fitness defects in vivo for P. mirabilis but do not affect colonization of E. coli during UTI. Similarly, the oxidative pentose phosphate pathway is required only for P. mirabilis in vivo. In contrast, gluconeogenesis is required only for E. coli fitness in vivo. The remarkable difference in central pathway utilization between E. coli and P. mirabilis during experimental UTI was also observed for TCA cycle mutants in sdhB, fumC, and frdA. The distinct in vivo requirements between these pathogens suggest E. coli and P. mirabilis are not direct competitors within host urinary tract nutritional niche. In support of this, we found that co-infection with E. coli and P. mirabilis wild-type strains enhanced bacterial colonization and persistence of both pathogens during UTI. Our results reveal that complementary utilization of central carbon metabolism facilitates polymicrobial disease and suggests microbial activity in vivo alters the host urinary tract nutritional niche.The human genitourinary tract is a common anatomical niche for polymicrobial infection and a leading site for the development of bacteremia and sepsis. Most uncomplicated, community-acquired urinary tract infections (UTI) are caused by  Escherichia coli, while another bacterium, Proteus mirabilis, is more likely to cause catheter-associated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent nutritional requirements despite growing in the same host environment. This result indicates that E. coli and P. mirabilis do not directly compete for nutrients during UTI. Indeed, we found that persistence of both pathogens is enhanced when they co-colonize the host. This work represents an important step toward understanding the basic nutritional requirements for two major pathogens that cause UTI and shows how mixed infections can change these requirements. Understanding how bacteria grow during infections is fundamental to ultimately uncover new ways to combat increasingly drug-resistant bacterial infections.The human urinary tract is a leading source for polymicrobial infections and for the development of bacteremia and sepsis. Treating these potentially dangerous infections have recently become more challenging due to the appearance of uropathogenic strains that are resistant to the many of the most commonly prescribed antibiotics. The majority of urinary tract infections (UTI) are caused by  Extraintestinal pathogenic E. coli require peptide import systems, the TCA cycle, and gluconeogenesis to consume amino acids and peptides in the urinary tract E. coli require pathways to catabolize multiple sugars available in the intestine Salmonella enterica serovar Typhimurium exploits the inflammatory response of the host that creates an alternative electron acceptor to allow the pathogen to respire and compete with anaerobic gut residents The recent revival of interest in the relationship between bacterial metabolism and host-pathogen interactions has deepened our understanding of pathogen colonization and growth Listeria monocytogenes, indicate that gluconeogenesis and uptake and catabolism of glycerol and dihydoxyacetone are required for bacterial replication in vivoL. monocytogenes intracellular replication Listeria in vivo. Shigella flexneri, which also replicates in the host cell cytosol, requires glycerol-3-phosphate as a carbon source E. coli (EIEC), which are genetically-related to S. flexneri, also use 3-carbon substrates as a carbon source in vivo. Because chorismate, GMP, and thymidylate synthesis have been found essential for S. flexneri replication in vivode novo synthesis of amino acids is extensive, it is believed that glycerol metabolism and anabolic pathways in this bacterium and EIEC may be important for bacteria that replicate within epithelial cells during intestinal infection.The contribution of central carbon pathways to pathogenesis has been shown for numerous intracellular and extracellular nutritional niches occupied by pathogens. Expression-based and genetic approaches using the model cytosolic pathogen, Mycobacterium tuberculosis that replicates intracellularly within phagocytes, utilizes fatty acids in addition to glycerol or glycerol-3-phosphate as a carbon source in macrophages in vivo carbon metabolism in M. tuberculosis is dependent on fatty acid catabolism and the glyoxylate shunt through the TCA cycle L. monocytogenes, S. flexneri, EIEC, and M. tuberculosis growth in vivo likely indicates that glycerol is a readily available carbon source inside host cells and extends Rolf Freter's nutrient-niche hypothesis S. enterica serovar Typhimurium because glucose import, glycolysis, and the oxidative TCA cycle are required for Salmonella to colonize the intestine and replicate within host phagocytes in vivo could reflect a Salmonella fitness adaptation for facing increased competition with a diversity of luminal gut anaerobes.E. coli, are able to occupy the host gastrointestinal tract, yet their nutritional requirements for carbon metabolism in vivo have key differences. Colonization studies using the streptomycin-treated mouse model in combination with transcriptional profiling during culture in mucus demonstrated that the ED pathway, and gluconate or other sugar acids, are required for intestinal growth of commensal E. coliin vivo requires the catabolism of up to six additional sugars E. coli, which solely utilizes glycolytic pathways for in vivo growth in vivo can alter preferred routes of carbon flux through the central pathways, introduces the notion that studying polymicrobial interactions during host colonization is essential to understand the relationship between bacterial metabolism and pathogenesis.Extracellular or luminal colonizers, including both commensal and pathogenic E. coli (ExPEC), it has been shown that D-serine metabolism and acetogenic growth are important during colonization of the urinary tract E. coli during extraintestinal infection, while glycolysis and the pentose phosphate pathway are dispensable E. coli has to synthesize sugars from amino acids (gluconeogenesis) while enzymes for sugar catabolism have no affect on fitness. Although less is known about the in vivo metabolism of Proteus mirabilis, another important urinary tract pathogen, it would be expected to have the same enzymatic requirements during infection. Attenuated strains of P. mirabilis have been identified with mutations in genes that encode proteins involved in gluconate and pyruvate metabolism, and in enzymes of the TCA cycle, using signature-tagged mutagenesis E. coliP. mirabilisin vivo between these uropathogens during experimental infection of the urinary tract. To better understand the relationship between the host nutritional niche and pathogen growth, we used defined mutants, each defective in specific metabolic pathways, to directly examine the in vivo metabolism for two bacterial pathogens that occupy the same host niche and likely have access to the same nutrients during infection.For extraintestinal pathogenic in vivo requirements for central pathways between these two pathogens during UTI by assessing the in vivo fitness of strains containing mutations in pgi, pfkA, tpiA, pykA, gnd, talB, edd, sdhB, fumC, frdA, and pckA, in both uropathogenic E. coli CFT073 and P. mirabilis HI4320  and pgi (phosphoglucose isomerase), mutants in irreversible glycolytic steps involving both 6-carbon  and 3-carbon  substrates were constructed and tested in competitive infections with the parental E. coli CFT073 strain. Using the well-established murine model of ascending infection E. coli to compete with wild-type CFT073 during experimental infection , all of the remaining P. mirabilis glycolysis mutants tested were out-competed by wild-type parental HI4320>1,000-fold (P<0.050) in both bladders and kidneys  does not negatively affect fitness during extraintestinal infection despite TalA being induced by CFT073 when cultured in human urine in vivo, an additional transaldolase mutant, transaldolase B , was constructed in E. coli CFT073. TalB is the major transaldolase in E. coli that transfers a three-carbon moiety from a C7 molecule to glyceraldehyde-3-P (C3) to form erythrose-4-P (C4) and fructose-P (C6). This stage of the pentose phosphate pathway is reversible, and thus, can be uncoupled from the oxidative decarboxylation reactions that produce NADPH. While loss of the major transaldolase, TalB, did not affect E. coli fitness during UTI   .E. coli and P. mirabilis requirement in the non-oxidative pentose phosphate pathway is the redundancy of transaldolase in E. coliP. mirabilis strains encode a single transaldolase enzyme , while E. coli strains have both TalA and TalB, which catalyze identical reactions for the cell. To determine if the lack of a fitness defect for the E. coli talB mutant    . AlthougP<0.050) .gnd) and a mutant defective in gluconate catabolism, 6-phosphoglyconate dehydrase (edd), were tested in competitive infections with the parental CFT073 wild-type strain. E. coli lacking either the oxidative pentose phosphate pathway (gnd), or the ED pathway (edd) were recovered from bladders and kidneys in numbers not significantly different from the wild-type strain (median CI = 1)   . SurprisP<0.003) .P. mirabilis edd transposon insertion as attenuated during experimental UTI edd was not confirmed by testing a ‘clean’ isogenic mutant strain in vivo. Despite this, it was reasonable to expect that, similar to the different requirement for glycolysis between E. coli and P. mirabilis during infection, P. mirabilis may require the capacity to metabolize gluconate via the Entner-Duodoroff pathway in vivo. Consistent with this, we found that in contrast to the findings with E. coli, the P. mirabilis edd mutant was significantly out-competed in both the bladders and kidneys by the parental HI4320 strain (P<0.020) during co-challenge infections when co-inoculated 1∶1 with wild-type ; fumC, and fumarate reductase; frdA were constructed and tested in competitive infections with wild-type E. coli CFT073 or P. mirabilis HI4320, respectively. While both E. coli and P. mirabilis required TCA cycle reactions for fitness in vivo, sdhB was required for fitness only during cystitis (bladder CFU) in E. coli (P. mirabilis (P>0.050). It is generally believed that the production of reduced FADH2 during the conversion of succinate to fumarate by succinate dehydrogenase is avoided during fermentation by modification of the TCA cycle to an incomplete reductive pathway where fumarate conversion to succinate by fumarate reductase replaces succinate dehydrogenase activity. The loss of FrdA resulted in a fitness defect for P. mirabilis during infection of both the bladder and kidneys (P>0.005) (E. coli frdA mutant colonization levels were indistinguishable from wild-type (median CI = 1.0) in the kidneys and significantly outcompeted the parental CFT073 strain>50-fold during acute cystitis (P = 0.024) , while the E. coli fumC mutant colonized the kidneys to similar levels as the parental CFT073 strain (CI = 0.94) and had a minor fitness defect in the bladder  (The aerobic tricarboxylic acid (TCA) cycle has been proposed to be required for  E. coli  and onlyirabilis  (P>0.050P>0.005) . In cont= 0.024) . Both E.= 0.031) .pckA) that generates phophoenolpyruvate and bypasses the irreversible glycolytic reaction catalyzed by pyruvate kinase (pykA). Mutation of pckA, which disrupts gluconeogenesis, resulted in a significant fitness defect for E. coli in both bladder and kidneys (P<0.005)   . Loss ofnfection . In suppP<0.005) .in vitro culture; pckA mutant strains in both CFT073 and HI4320 backgrounds were indistinguishable from parental strains in LB medium and defined medium containing glucose  to assimilate nitrogen P. mirabilis assimilates nitrogen, via glutamate dehydrogenase (Gdh) P. mirabilis requiring glycolysis, pentose phosphate pathway, and the ED pathway; while, on the other hand, the exact same mutations in E. coli have no affect on fitness during UTI. Alternatively, our findings raise the possibility that E. coli and P. mirabilis could reside in different cellular compartments. For example, it has been shown that E. coli can reside intracellularly during acute infection P. mirabilis can invade cultured cells P. mirabilis spends a significant portion of the acute infection within host cells.Our finding that E. coli during UTI. In our previous work we assessed glycolysis by studying a mutation in the gene that encodes triose phosphate isomerase E. coli and P. mirabilis. These enzymes perform irreversible steps in glycolysis, thus by testing these mutations during UTI it is now clear that glycolysis is dispensible for E. coli and is required for P. mirabilis fitness in vivo. That glycolysis is required for P. mirabilis but not E. coli raises the possibility that sugars become available within the urinary tract during Proteus infection. That sugars become available when P. mirabilis colonizes the urinary tract would also allow for enhanced colonization of E. coli because sugars available during co-infection might increase growth of E. coli over numbers it would normally reach by solely consuming amino acids. This could also explain why gnd mutant E. coli displayed a fitness defect only when co-colonized with P. mirabilis.In addition to finding a remarkable difference in central metabolism requirements between two pathogens that infect the urinary tract, these findings have validated that glycolysis is dispensable for E. coli fitness during UTI E. coli as predicted by our earlier study. In contrast, fumarate reductase mutants in P. mirabilis are out-competed by wild-type. These data show that the branched, reductive TCA pathway is not important for E. coli to grow within the urinary tract, supporting the notion that the urinary tract is moderately oxygenated P. mirabilis fitness during UTI suggests an anaerobic environment does exist in the host urinary tract. In addition, because sensing oxygen depletion has been shown to be important for E. coli fitness during UTI E. coli due to the presence of alternative energy pathways like Ni-Fe hydrogenases and nitrate reductase, these and other redox enzymes are part of the large repertoire of electron transfer components available for the highly modular respiratory chains that can be assembled in E. coliE. coli during UTI.It has also been shown that the oxidative TCA cycle is important for E. coli and P. mirabilisE. coli virulence E. coli and P. mirabilis may not directly compete for nutrients during colonization of the urinary tract and led us to hypothesize that co-inoculation of E. coli and P. mirabilis may lead to enhanced colonization levels during experimental UTI. Indeed, we found that colonization was enhanced for both E. coli and P. mirabilis. The benefit of polymicrobial infection was more apparent for E. coli over time. In addition to supporting that E. coli and P. mirabilis may not directly compete for resources during UTI, these findings could alternatively suggest that some activity of one or both pathogenic bacteria may alter the host niche in such a way that facilitates bacterial replication. In support of this, we observed that P. mirabilis causes more tissue damage and larger induction of the pro-inflammatory cytokines IL-1α, IL-β, IL-6, and G-CSF than does E. coli in independent infection, while a mixed infection appears more similar to E. coli alone  medium. For growth experiments, wild-type and mutant strains of E. coli and P. mirabilis were cultured in MOPS defined medium 600 was recorded every hour.P. mirabilis HI4320 mutants (E. coli CFT073 deletion mutants (E. coli mutants was carried out by PCR using primers flanking the target gene sequence and comparing product size to wild-type PCR product size. When size differences were negligible PCR products were digested with a restriction enzyme (New England Biolabs). mutants  were gen mutants  were conin vitro complementation, the tpiA gene was amplified from P. mirabilis genomic DNA using Easy-A high fidelity polymerase (Stratagene) and independently cloned into pGEN-MCS tpiA were verified by DNA sequence analysis prior to electroporation into the P. mirabilis tpiA mutant strain and E. coli tpiA mutant strain.For 7 or 2×108 CFU) per mouse using a sterile polyethylene catheter (I.D. 0.28 mm × O.D. 0.61 mm) connected to an infusion pump (Harvard Apparatus). For in vivo co-challenges, a suspension containing 5×107 CFU of a 1∶1 ratio of P. mirabilis HI4320 and P. mirabilis kanamycin-resistant mutant in LB medium or a suspension containing 2×108 CFU of a 1∶1 ratio of E. coli CFT073 and E. coli antibiotic-resistant mutant in PBS. For independent infections, the respective P. mirabilis or E. coli suspensions contained only the wild-type strain. Input CFU/ml was determined by plating serial dilutions  of each inoculum onto low salt (0.5 g NaCl/L) LB agar, to prevent P. mirabilis swarming, with and without antibiotic. For experiments with P. mirabilis, low salt LB agar (0.5 g NaCl/L) was used to prevent swarming. Infected mice were euthanized 48 h or 7 d post infection, bladder and kidneys were aseptically removed, weighed, and homogenized  in 3 ml PBS, and appropriate dilutions were spiral plated on LB agar with and without antibiotic to determine the output CFU/g of tissue. Viable counts were enumerated using QCount software  and CFU from antibiotic-containing medium (mutant CFU) were subtracted from the total CFU from plates lacking antibiotic to determine the number of wild-type bacteria. For co-challenge experiments, competitive indices (CI) were calculated by dividing the ratio of the CFU of mutant to wild-type recovered from each mouse following infection by the ratio of the CFU of mutant to the CFU of wild-type present in the input. CI data were log-transformed and analyzed by the Wilcoxon signed-rank test to determine statistically significant differences in colonization . A CI>1 indicates that the mutant out-competes the wild-type strain and a CI<1 indicates that the mutant is out-competed by the wild-type strain. For independent infections the Mann-Whitney test was used to determine statistically significant differences in colonization .The CBA mouse model of ascending UTI in vivo for bacteria during polymicrobial infection was determined by co-inoculating UPEC CFT073 and P. mirabilis HI4320 strains and deletion mutants into the same female CBA mice as described previously with the following modification. For polymicrobial co-challenge infections, a 1∶1 (v/v) mixture was prepared containing 2.5×107 CFU of P. mirabilis HI4320 in LB medium and 108 CFU E. coli CFT073 in PBS. Competitive indices were calculated as described above. For polymicrobial infections containing only wild-type strains, the CFU/g tissue were determined following plating of serial dilutions on low salt (0.5 g NaCl/L) LB agar with and without tetracycline; P. mirabilis is intrinsically tetracycline-resistant. CFU from tetracycline agar plates, which represent P. mirabilis, were subtracted from total CFU recovered on LB agar without antibiotics to determine CFU/g for E. coli. For quantification of bacteria recovered from a polymicrobial infection with a wild-type strain and a heterologous kanamycin-resistant mutant strain, CFU on LB agar containing kanamycin (mutant) were subtracted from total CFU recovered on LB without antibiotics, to determine wild-type CFU/g tissue. Tetracycline was used to enumerate bacterial colonization levels following polymicrobial infection with heterologous kanamycin-resistant strains.The relative fitness All animal experiments were performed in accordance to the protocol (08999-3) approved by the University Committee on Use and Care of Animals at the University of Michigan. This protocol is in complete compliance with the guidelines for humane use and care of laboratory animals mandated by the National Institutes of Health.S1 FigIn vitro growth of pentose phosphate and Entner-Doudoroff pathway mutants. Growth of  UPEC CFT073 and  P. mirabilis HI4320 wild-type strains and mutants in: gnd, 6-phosphogluconate dehydrogenase; talB, transaldolase; and edd, 6-phosphogluconate dehydratase in LB medium  or defined medium containing 0.2% glucose  as the carbon source. A representative growth curve is shown for each panel.(TIF)Click here for additional data file.S2 FigIn vivo contribution of arginine and serine biosynthesis.  Competitive indices (CI) were determined following co-challenge infections with female CBA/J mice with a 1∶1 ratio of either wild-type (A) E. coli CFT073 or (B) P. mirabilis HI4320 and their respective mutants in: argG, argininosuccinate dehydrogenase and serA, D-3-phosphglycerate dehydrogenase. UPEC was recovered at 48 h post-inoculation. P. mirabilis was recovered at 7 d post-inoculation. Each dot represents bladder (closed symbols) and kidneys (open symbols) from an individual animal. Bars indicate the median CI. Significant differences in colonization (*P<0.05) were determined by the Wilcoxon signed-rank test. A CI<1 indicates a fitness defect. Growth of P. mirabilis HI4320 wild-type strain and amino acid auxotroph mutants in (C) LB medium and (D) defined medium containing 0.2% glucose with or without 10 mM of the indicated amino acid.(TIF)Click here for additional data file.S3 FigIn vitro co-culture of wild-type E. coli CFT073 and P. mirabilis HI4320. A 1∶1 ratio containing 106 CFU/ml of each strain was used to inoculate LB medium (solid symbols) and minimal salts medium containing 0.2% glucose (open symbols). Co-cultures were incubated at 37°C with agitation for 24 h; log CFU/ml were determined following plating of serial dilutions on LB agar with and without tetracycline. CFU from tetracycline-containing plates (P. mirabilis) were subtracted from total CFU recovered on LB agar without antibiotics to determine CFU/ml for E. coli (TetS).(TIF)Click here for additional data file.S4 FigE. coli colonization of the urinary tract is enhanced following pre-colonization by P. mirabilis during sequential infection. Colonization levels of (A) E. coli CFT073 or (B) P. mirabilis HI4320 following sequential in vivo co-infections. On day zero (0) female CBA/J mice were infected with either wild-type strain E. coli CFT073 or P. mirabilis HI4320. Following 2 days (2) mice were inoculated with the other wild-type species. Mice were euthanized and organs collected 48 h following the secondary infection. Each dot represents log10 CFU/g tissue of bladder or kidneys from an individual animal. The bars indicate median log10 CFU for bladder and kidneys. CFU/ml were determined following plating of serial dilutions on LB agar with and without tetracycline (15 µg/ml). CFU from Tet plates (P. mirabilis) was subtracted from total CFU recovered on LB agar without antibiotics to determine CFU/ml for E. coli (TetS). P-values indicated on the graph were determined by the Mann-Whitney test.(TIF)Click here for additional data file.S5 FigUrinary tract pathology and urinary proinflammatory cytokine production during mono-species and polymicrobial infection. Groups of 5 mice (n = 15) were inoculated with E. coli CFT073 alone, P. mirabilis HI4320 alone, or a 1∶1 mixture of both E. coli and P. mirabilis. (A) Inflammation and lesions were scored for pyelonephritis, peripelvic inflammation, and cystitis Tissue pathology was scored from 0–3, with 3 being most severe. Severity was determined by assessing for the presence of PMNs and lymphocytes, focal aggregates of PMNs or lymphocytes, edema, and transmural inflammation. (B) Urine was collected from all 15 mice before inoculation (uninfected control) and from infected mice at 24, 48, 72, and 96 h post-inoculation. Urine from each group were pooled and used to analyze a panel of 12 pro-inflammatory cytokines by ELISA.(TIF)Click here for additional data file.S1 TableBacterial strains used in this study.(DOCX)Click here for additional data file.


Salmonella enterica serotype Typhi (S. Typhi), is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some S. Typhi isolates exhibit over-expression of Vi polysaccharide, which masks clinical detection of LPS O-antigen. This variation in Vi polysaccharide and O-antigen display (VW variation) has been observed since the initial studies of S. Typhi. We have reported that the status of the rpoS gene is responsible for this phenomenon. We review the regulatory network of the Vi polysaccharide, linking osmolarity and RpoS expression. Also, we discuss how this may impact live attenuated Salmonella vaccine development.The synthesis of Vi polysaccharide, a major virulence determinant in  Salmonella enterica serotype Typhi (hereafter S. Typhi) is a facultative intracellular pathogen that causes typhoid fever exclusively in humans and is among the most costly of human infections in terms of both morbidity and mortality [S. Typhi is different from that of other serovars of Salmonella, and in this regard S. Typhi produces the virulence capsular polysaccharide (Vi), which is an important virulence determinant during infection [S. Typhi in the gallbladder are found to express Vi polysaccharide antigen [+) strains were shown to be more virulent than + strains are resistant to complement-mediated killing and phagocytosis [+ strains, but not Vi− mutant strains, can multiply in the human macrophage cell line THP-1 and in the mouse macrophage-like cell line J774.1 [ortality . The mecnfection . Vi polynfection ,4 and hanfection . Virtual antigen ,7,8. Vi lunteers ; Vi+ strocytosis  and survocytosis . In addie J774.1 .S. Typhi mutants that constitutively express high levels of Vi [S. Typhi pathogenesis, but also allow us to improve Vi antigen production and therefore provide a less expensive subunit vaccine.The Vi capsular polysaccharide is a protective antigen, with the majority of the antibody response directed toward the O-acetyl groups ,12. A Vils of Vi . TherefoviaB locus) are located in a 133.5 kb chromosomal region called Salmonella pathogenicity island 7  and contains genes encoding known pathogenicity determinants, including SopE [S. Typhi revealed significant synteny with clusters of genes from a variety of soil saprophytic bacteria and phytobacteria, raising the possibility that SPI-7 and viaB may have originated from soil sources [The genes required for Vi biosynthesis  [ompR-envZ (ompB locus) [tviA promoter [tviA promoter terminates in the tviA-tviB intergenic region, probably at a putative hairpin structure identified in this region [The regulation of Vi polysaccharide synthesis is complex and may play a significant role in maintaining appropriate levels of Vi production in the different host environments encountered by A locus)  and ompRB locus) , which bpromoter  and intepromoter , 28, 29.S. Typhi Ty2 grown in media with medium osmolarity , optimal for Vi polysaccharide synthesis, showed that Vi polysaccharide reduced TLR-dependent IL-8 production in human colonic tissue explants, suggesting that the scarcity of neutrophils in intestinal infiltrations of typhoid fever patients is due to the Vi polysaccharide [flhDC, which is apparently required for activation of iagA (hilA), invF and sipB (encoding proteins involved in cell invasion) [iagA, invF, and sipB is markedly increased and the transcription of genes involved in Vi biosynthesis is markedly decreased [S. Typhi is more invasive into epithelial cells but less resistant to killing by macrophages [S. Typhi is a negative factor for invasion but a positive factor for surviving and multiplying inside macrophages [et al. showed that at ≥ 300 mM NaCl, but not at 10 mM NaCl, S. Typhi GIFU1007 did not express Vi antigen and exhibited a high invasion index in epithelial cells together with high secretion of SipC protein [Vi is expressed preferentially at low and medium osmolarities and often masks the LPS O-antigen ,31. Stra mM NaCl  but are  mM NaCl . Low to  mM NaCl . It is p mM NaCl . Recent ccharide . In addinvasion) , 34. Howecreased . Under trophages . Thereforophages . In keep protein .Salmonella somatic D1 antiserum cannot be demonstrated until the bacterial cells are boiled to remove the Vi polysaccharide [The synthesis of Vi antigen increases as the osmolarity decreases , maskingccharide .S. Typhi, variation in Vi and O-antigen detection has been observed. S. Typhi strains non-agglutinable with O-antisera and agglutinable only with Vi antisera, are called V form while S. Typhi strains that lack the Vi antigen and agglutinate only with O-antisera are called W form [S. Typhi, were the VW form.Starting from the initial studies on d W form . Observad W form  and confd W form  demonstrd W form . Coincidd W form , and sind W form ,40,41,42Salmonella, the rpoS gene encodes an alternative sigma factor (σs/RpoS) that is the master regulator in the general stress response and is required for survival under extreme conditions, including osmotic and oxidative stress, transition to stationary phase, acid shock, [S. Typhimurium [S. Typhimurium virulence plasmid genes, spvRABCD [S. Typhi does not contain a virulence plasmid and the role of rpoS in the virulence of this serotype has not been rigorously studied. However, rpoS might also contribute to the virulence of this serotype because RpoS− strains of S. Typhi are less cytotoxic than RpoS+ strains, but RpoS− strains survive better inside resting THP-1 macrophages without apoptosis induction [rpoS gene [In d shock, ,44 and fhimurium , 48,49. spvRABCD , 47. In nduction . Recent poS gene .rpoS through the RcsB-RprA pathway [rpoS genotypes, in media with different osmolarities, for levels of Vi antigen synthesis. RpoS+ strains grown at osmolarities less than 676 mosmol  showed enotypes . Pickard  and ISP1820 (RpoS+), but Ty2 produced more Vi than ISP1820 at all osmolarities tested . These results indicate that the RpoS allelic state is responsible for the VW and V variation in S. Typhi. RpoS− strains over-express the Vi polysaccharide without RpoS regulation, leading to a permanent V form. RpoS+ strains exhibit both forms, the V form during the early exponential growth, when RpoS is not expressed or expressed at low levels, and the VW form, when RpoS is expressed. The W form (Vi−) can be caused by spontaneous deletion of SPI-7, where the viaB loci is located [RpoS is a key factor in the stress response during the transition from the exponential growth phase to the stationary growth phase . Very litability ,57. To dry phase , althougry phase . There w located .− and Vi− [S. Typhi Ty2 [rpoS mutation could affect the immunogenicity in recombinant vaccines [−) have yielded poor results when used as a live recombinant S. Typhi vaccine (RAStyVs) expressing protective antigens from a diversity of pathogens [rpoS mutation rather than Vi antigen deletion. On the other hand, over-expression of Vi antigen in Ty2 could decrease adherence to and invasion into intestinal tissues necessary to colonise more internal lymphoid effector tissues [S. Typhimurium, it has been demonstrated that chromosomal RpoS-regulated genes are necessary for invasion into and colonisation of the gut-associated lymphoid tissue (GALT) [−S. Typhimurium mutants exhibit diminished immunogenicity [S. Typhimurium virulence plasmid appear to play no role in this effect [The live typhoid vaccine Ty21a, which is an RpoS, GalE and Vi−  derivatiyphi Ty2 , has beeyphi Ty2 ,60,61,62vaccines . In factathogens ,67,68,69 tissues . In S. Te (GALT) . In accogenicity ,71. The s effect ,71.S. Typhi ISP1820 (RpoS+) seems to be more virulent in humans than S. Typhi Ty2 (RpoS−) [S. Typhi CVD906, a live vaccine, derived from ISP1820 with deletion mutations caused fever and other adverse reactions in humans in aroC and aroD [S. Typhi CVD908 Ty2 ΔaroC ΔaroD vaccine strain, which did not cause any adverse effects [+S. Typhi, with or without ability to produce the Vi capsular antigen, might be superior to RpoS− strains as a vector in the development of recombinant attenuated Salmonella vaccines for humans [S. Typhi Ty2 vaccine constructions. We are currently testing this hypothesis in human volunteers.Furthermore,  (RpoS−) . In concand aroD , is highand aroD ,74. This effects . These rr humans ,76, alth


Rhizobium leguminosarum bv. trifolii is a soil-inhabiting bacterium that has the capacity to be an effective N2-fixing microsymbiont of Trifolium (clover) species. R. leguminosarum bv. trifolii strain WSM1689 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Trifolium uniflorum collected on the edge of a valley 6 km from Eggares on the Greek Island of Naxos. Although WSM1689 is capable of highly effective N2-fixation with T. uniflorum, it is either unable to nodulate or unable to fix N2 with a wide range of both perennial and annual clovers originating from Europe, North America and Africa. WSM1689 therefore possesses a very narrow host range for effective N2 fixation and can thus play a valuable role in determining the geographic and phenological barriers to symbiotic performance in the genus Trifolium. Here we describe the features of R. leguminosarum bv. trifolii strain WSM1689, together with the complete genome sequence and its annotation. The 6,903,379 bp genome contains 6,709 protein-coding genes and 89 RNA-only encoding genes. This multipartite genome contains six distinct replicons; a chromosome of size 4,854,518 bp and five plasmids of size 667,306, 518,052, 341,391, 262,704 and 259,408 bp. This rhizobial genome is one of 20 sequenced as part of a DOE Joint Genome Institute 2010 Community Sequencing Program. BNF is performed only by a specialized subset of Bacteria and Archaea that possess the necessary cellular machinery to enzymatically reduce N2 into NH3. Some of these bacteria (termed rhizobia or root nodule bacteria) have evolved non-obligatory symbiotic relationships with legumes whereby the bacteria receive a carbon source from the plant and in return supply fixed N to the host [The nitrogen (N) cycle is one of the most important biogeochemical processes underpinning the existence of life on Earth. A key step in this cycle is to convert relatively inert atmospheric dinitrogen (Nthe host . HarnessTrifolium (clover). The natural distribution of these species spans three centers of diversity, with an estimated 28% of species in the Americas, 57% in Eurasia and 15% in sub-Saharan Africa [T. repens (white clover), T. pratense (red clover), T. fragiferum (strawberry clover) and T. hybridum . While clovers are known to form N2-fixing symbiotic associations with Rhizobium leguminosarum bv. trifolii, there exists wide variation in symbiotic compatibility across different strains and hosts from ineffective (non-N2-fixing) nodulation to fully effective N2-fixing partnerships.Some of the most widely cultivated pasture legumes are members of the legume genus n Africa . Approxin Africa . GloballRhizobium leguminosarum bv. trifolii strain WSM1689 was isolated in 1995 from a nodule of the perennial clover Trifolium uniflorum collected on the edge of a valley 6 km from Eggares on the Greek Island of Naxos. T. uniflorum is one of small number of perennial Trifolium spp. found in the dry, Mediterranean basin. While WSM1689 has been shown to be either ineffective or unable to nodulate a range of annual and perennial Trifolium sp., it is a highly effective N2-fixing microsymbiont of T. uniflorum [R. leguminosarum bv. trifolii WSM1689 has a very narrow host range and thus represents a good isolate to study the genetic basis of symbiotic specificity. The availability of this sequence data also complements the already published genomes of the clover-nodulating R. leguminosarum bv. trifolii WSM1325 [R. leguminosarum bv. trifolii strain WSM1689 together with the description of the complete genome sequence and its annotation.niflorum . TherefoR. leguminosarum bv. trifolii strain WSM1689 is a motile, non-sporulating, non-encapsulated, Gram-negative rod in the order Rhizobiales of the class Alphaproteobacteria. The rod-shaped form varies in size with dimensions of approximately 0.25-0.5 μm in width and 2.0 μm in length (n length  Right. MR. leguminosarum bv. trifolii strain WSM1689 in a 16S rRNA gene sequence based tree. This strain shares 100% (1362/1362 bp) sequence identity to the 16S rRNA gene of R. leguminosarum bv. trifolii strain WSM1325 [R. leguminosarum bv. trifolii strain WSM2304 [R. leguminosarum bv. trifolii WSM1689 is a highly effective microsymbiont of the perennial Eurasian clover Trifolium uniflorum (T. fragiferum and forms white ineffective (Fix-) nodules with other perennial and annual clovers of Eurasian origin. Moreover, WSM1689 is either Nod- or Fix- on clovers of North American or African origin. Therefore, WSM1689 is unusual in having an extremely narrow clover host range for the establishment of effective N2-fixing symbiosis.niflorum . In contThis organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database  and a fiRhizobium leguminosarum bv. trifolii strain WSM1689 was grown to mid logarithmic phase in TY rich medium on a gyratory shaker at 28°C [ at 28°C . DNA was at 28°C .Rhizobium leguminosarum bv. trifolii strain WSM1689 was sequenced at the Joint Genome Institute (JGI) using a combination of Illumina [The genome of Illumina  and 454 Illumina . An IlluIllumina . The iniIllumina , and theIllumina -36 was uIllumina , or sequGenes were identified using Prodigal  as part The genome is 6,903,379 nucleotides with 60.94% GC content , and com


Ascochyta rabiei is a necrotrophic fungus that causes devastating blight disease of chickpea (Cicer arietinum). Here, we report a 34.6 megabase draft genome assembly of A. rabiei. The genome assembly covered more than 99% of the gene space and 4,259 simple sequence repeats were identified in the assembly. A total of 10,596 high confidence protein-coding genes were predicted which includes a large and diverse inventory of secretory proteins, transporters and primary and secondary metabolism enzymes reflecting the necrotrophic lifestyle of A. rabiei. A wide range of genes encoding carbohydrate-active enzymes capable for degradation of complex polysaccharides were also identified. Comprehensive analysis predicted a set of 758 secretory proteins including both classical and non-classical secreted proteins. Several of these predicted secretory proteins showed high cysteine content and numerous tandem repeats. Together, our analyses would broadly expand our knowledge and offer insights into the pathogenesis and necrotrophic lifestyle of fungal phytopathogens.Constant evolutionary pressure acting on pathogens refines their molecular strategies to attain successful pathogenesis. Recent studies have shown that pathogenicity mechanisms of necrotrophic fungi are far more intricate than earlier evaluated. However, only a few studies have explored necrotrophic fungal pathogens.  Cicer arietinum L.), an important high-protein source, is an annual legume crop grown worldwide. The chickpea crop yield suffers primarily from Ascochyta blight (AB) that is caused by the necrotrophic ascomycete fungus Ascochyta rabiei (Pass.) Labr. [teleomorph: Didymella rabiei (Kovatsch.) Arx], causing up to 100% yield lossA. rabiei is still not clearly understood ever after several pathological and molecular studies. Therefore, comprehensive information on the biology and survival of A. rabiei is a prerequisite to develop more effective disease management strategies.Chickpea  repertoire of A. rabiei was identified and an in-depth comparison was performed with CAZymes of other representative necrotrophic and biotrophic fungi. The effector repertoire of A. rabiei was predicted and secretome annotation was performed to gain maximum knowledge about effectors related to necrotrophy. The predicted effector proteins of A. rabiei would be useful in identifying their resistant counterparts in chickpea and for designing efficient strategies against this devastating but poorly characterized pathogen.With this objective, we described the A. rabiei (ITCC No. 4638) for sequencing. This isolate was identified as mating type 2 (MAT1–2) using MAT locus-specific primers  were also identified, which indicates that the assembly covered more than 99% of the gene space. The average gene density in the A. rabiei genome was 305 genes per Mb  of A. rabiei predicted proteins had orthologs in these three fungal species, while 693 (6.5%) predicted proteins were unique , indicating it as the nearest species among the selected fungi  also suggested that A. rabiei was closely related to S. nodorum (The predicted proteome of e unique . Interese unique . A largeed fungi . Phyloge nodorum .A. rabiei proteome were compared with those present in the three most closely related Dothideomycetes fungi (A. rabiei proteome unveiled high abundance of major facilitator superfamily (MFS) transporters, protein kinases, short-chain dehydrogenases/reductase family, zinc cluster domains and sugar transporters. In contrast, cytochrome P450 family proteins were significantly less abundant in A. rabiei. Moreover, unlike C. heterostrophus and S. nodorum but similar to P. tritici-repentis, heterokaryon incompatibility protein (HET) in A. rabei was found in low abundance. The protein-protein interaction  and FAD binding domains (PF01565) were found considerably less in A. rabiei than in the other three fungi, suggesting variation in A. rabiei genome from its closely related Dothideomycetes fungi.Pfam annotation was assigned to 7,118 genes (67.17%) . The Pfaes fungi . The A. A. rabiei, C. heterostrophus and P. tritici-repentis) and biotrophic fungi . OrthoMCL analysis showed that 1,458 and 112 proteins were orthologous among the necrotrophic and biotrophic fungi, respectively  in the genome suggested that A. rabiei might possess a broad specificity for utilizing nutrients from host plants.Transporters involved in nutrient uptake and re-allocation play multiple vital roles in growth and development. In total, 821 transporter proteins belonging to 90 families were identified in assembly . The higassembly . Among aA. rabiei genome assembly, 26 clusters harboring putative secondary metabolite genes were identified, suggesting possible production of biologically active compounds  showed presence of Pfam protein domain that matched with at least one of the CAZyme families. These putative CAZymes included 58 families of glycoside hydrolases (GHs), 40 families of glycosyl transferases (GTs), 8 families of carbohydrate esterases (CEs), 18 families of carbohydrate-binding modules (CBMs), 9 families of auxiliary activities (AAs) and only 3 families of polysaccharide lyases (PLs)  . Among aes (PLs) . The rel CAZymes , particu CAZymes . FurtherA. rabiei, genome-wide BLAST analyses using the protein sequences in the Pathogen-Host Interaction Database (PHI database)A. rabiei were predicted to be orthologous to PHI genes  including 538 classical and 220 non-classical secreted proteins. Interestingly, 52 classical and 20 non-classical secretory proteins were present among 693 proteins unique in A. rabiei , biological process (321) and cellular component (71)  . Fifty-fent (71) . Under bent (71) . Within A. rabiei secreted CAZymes. Very high prevalence of GHs, CEs and AAs, which are required for degradation of the structures of plant cells was observed. In contrast, CBMs that functions in modification of the fungal cell wall for growth or protection from host-defenses were also in abundance. The most prevalent GHs CAZyme class was GH28 and GH43 which represented polygalacturonase and xylanase, respectively  were annotated with the CAZyme database . The repectively . Polygalductases . TherefoA. rabiei putative effectors, 323 putative effectors (lacking CAZymes and known domain proteins) were searched for the presence of orthologs in closely related C. heterostrophus, P. tritici-repentis, and S. nodorum . Out of 323, only 148 putative effectors had their orthologs in at least one of the three fungi whereas, 175 putative effectors were unique to A. rabiei suggesting that effectors are less conserved in nature A. rabiei to counteract the adverse effects of proliferating TEs. The higher occurrence of CpT ↔ TpT transitions and relatively lower frequency of CpA ↔ TpA mutations were observed as prominent features. However, CpT ↔ TpT substitutions do not code for nonsense mutations, suggesting RIP resistance in the tandem repeats. A similar phenomenon has been observed in M. grisea accompanying CpA-targeted mutation in RIP-affected sequencesNecrotrophic fungi are drawing more attention due to their unique lifestyle and devastating nature. However, their strategies for pathogenesis are difficult to understand. The A. rabiei genome to the necrotrophic wheat pathogen S. nodorum  compared to those in the genomes of the necrotrophic fungal pathogens P. tritici-repentis S. nodorum C. sativus A. rabiei or a result of the stringent methodology of gene prediction adopted to minimize redundant genes. Particularly, four categories of functional proteins were drastically reduced in A. rabiei. First, the WD40, ankyrin repeat, BTB  and other domains that are involved in protein-protein interactions were significantly fewer. This indicates that in A. rabiei, lower abundances of a few families of proteins involved in protein-protein interactions, scaffolding proteins and enzymes with varying co-factors suggested broader specificity of these families for their downstream proteins to perform necessary biological functions despite of their low abundance. Secondly, few classes of enzymes with varying co-factors  were less in number and suggested that these enzymes classes might have broad specificity for their substrates in A. rabiei for carrying out important enzymatic reactions essential for its life cycle. Third, the HETs were also significantly fewer compared to those in C. heterostrophus and S. nodorum genomes. In filamentous fungi, genetic differences in HETs are known to limit viable heterokaryon formation between two different WT strainsA. rabiei indicated a higher tendency to fuse with dissimilar WT strains, leading to a higher probability of horizontal transfer of genetic elements. Fourth, the CYPs that are involved in detoxification of phytoalexin repertoires of host plantsA. rabiei due to lower adaptation. However, glutathione S-transferases (GSTs) were higher in abundance that functions in the detoxification of xenobiotic substrate, which may further aid in resistance against fungicides under field conditions. In addition, A. rabiei had a large inventory of CAZymes with a high capacity to degrade cellulose, pectin and xylan. These results correlated with the necrotrophic lifestyle of A. rabiei, where nutrition is obtained by degrading plant tissue.The comparative genome analyses suggested maximum closeness of the  nodorum . In addidorum 12,3C. sativA. rabiei effector reservoir included homologs of extracellular cutinase Pbc1 of Pyrenopeziza brassicaeU. maydisM. oryzaeA. rabiei secretome consists of diverse proteins that function in an organised manner to suppress different aspects of plant immunity for causing disease successfully.Secretory proteins play crucial roles during early colonization and pathogenesis. Of 758 predicted secretory proteins, 546 were non-CAZymes and might be potential effector candidates. GO analysis showed that the majority of the secretory proteins are likely to respond to oxidative stress. These proteins may be secreted to counteract host-generated oxidative stress. The pathogenicity related proteins of A. rabiei a successful necrotrophic pathogen. Detailed comparative genomics studies may provide unexpected new insights into biological phenomena of general interest. Functional characterization of potential effector candidates is a prerequisite for determining their roles in pathogenesis. Such studies will provide further insight and help in designing strategies to control this devastating disease and other necrotrophic fungal diseases.In summary, the present study has unlocked new prospects for the comprehensive genomic study of a variety of biological processes that make A. rabiei isolate ArD2 (Indian Type Culture Collection No. 4638) was obtained from the Division of Plant Pathology, Indian Agricultural Research Institute  and was used for whole genome sequencing. ArD2 is a highly virulent isolate with pinkish black spores. Vegetative mycelia were grown on potato dextrose agar  for 20 days or in potato dextrose broth  for 5 days at 22 °C in an incubator shaker at 120 rpm in the dark. Mycelial balls were harvested, and then total DNA was isolated using a DNeasy Plant Maxi kit (Qiagen) as per the manufacturer’s instructions.The A. rabiei was sequenced using an Illumina HiSeq1000 sequencing platform. The DNA libraries of 200, 300, 500 and 200–500 bp inserts, along with a mate-pair library of 5 kb insert size, were generated for sequencing purposes. These libraries were then paired-end sequenced. The reads obtained from the Illumina sequencing were trimmed using FASTX-toolkit (v0.0.13.2) and bases having a quality score less than 20 were removed from both ends. After trimming, the reads with lengths <70 bp were discarded. The draft genome was assembled with the help of ABySSk-mer 23, and the gaps were filled using GapFillerThe genome of de novo repetitive elements in the A. rabiei genome. It generated a library of 278 repetitive families with l-mer size 15, which included transposable elements (TEs) and dispersed duplicated sequences. This library was then filtered using following parameters: 1) Predicted repeats were aligned to genome assembly via BLASTN and hits were discarded if alignment length was <50 bp; 2) Repeats with frequency <5 in the genome were removed, and 3) Those repeats were also discarded for which significant hits to known proteins were found in Uniprot, except the ones showing hits to the known TEs. The resultant 155 consensus sequences were classified using TEclasshttp://www.girinst.org/repbase/index.html) by TBLASTX search. Then, the A. rabiei genome assembly was masked with 155 repetitive families using RepeatMaskerRepeatScouthttp://pgrc.ipk-gatersleben.de/misa/download/misa.pl) with default parameters. The default parameters used were: minimum SSR motif length of 10 bp and repeat length of mono-10, di-6, tri-5, tetra-5, penta-5, and hexa-5; the maximum size of interruption allowed between two different SSRs in a compound sequence was 100 bp.A high-throughput SSR search to identify mono- to hexanucleotide SSR motifs was performed using MIcroSAtellite identification tool (MISA)  were selected as default gene models. This resulted in prediction of 10,708, 11,293 and 10,843 protein coding genes, respectively. Altogether 51,850 genes predicted from all the three programs were used to retrain AUGUSTUS (with parameters from C. sativus as default gene model) and then new genes were predicted. Additionally, annotated proteins from S. nodorum, C. sativus and P. tritici-repentis were mapped onto the genome of A. rabiei using Exonerate: protein2genome. The resultant mapped genes from Exonerate were mapped back to the genes predicted by the retrained AUGUSTUS and only the genes which could be mapped were selected.Protein-coding genes in the A. rabiei. The BLASTP search was carried out against the single-copy families that contribute 246 single copy genes from all 21 species available in the FUNYBASEIn order to evaluate the genome completeness, the highly conserved single or low copy genes were searched in the predicted proteins of A. rabiei predicted genes, BLASTX search against NCBI non-redundant database was performed with cut-off E-values of ≤ 1e-5 and identity ≥40%. Gene ontology (GO) analysis was carried out using BLAST2GOhttp://pfam.xfam.org/). For CAZymes prediction, CAZymes Analysis Toolkit (CAT)For functional annotation of http://tree.bio.ed.ac.uk/software/figtree/).The phylogeny was performed using amino acid sequences of actin (ACT), beta-tubulin (BTUB), translation elongation factor-1 alpha (TEF1) and NAD-dependent glycerol-3-phosphate dehydrogenase (GPD). Protein sequences were downloaded from GenBank. The amino acid sequences were aligned in T-REXA. rabiei, S. nodorum, C. heterostrophus and P.tritici-repentis were identified with the help of OrthoMCLThe orthologous groups among A. rabiei was analyzed in SignalP v4.1 for prediction of the secretory signal peptide. The protein sequences lacking the signal peptide  were analyzed by SecretomeP v1.0The 10,596 protein set of A. rabiei secretome. An annotation method “based on association rules between CAZy families and Pfam domains” was used with an E-value threshold of 0.01, a bitscore threshold of 55 and rule support level 40. The predicted secretory proteins that could not be annotated by any of the above approaches were analyzed for the presence of characteristic features of effector proteins. In such proteins, high cysteine residue content and tandem repeats were examined. The number of cysteine residues was identified using Perl script. Protein internal repeats were predicted using T-Reks (http://bioinfo.montp.cnrs.fr/?r=t-reks/). The in planta localization of mature effector proteins were predicted by WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html). WoLF PSORT analysis was performed using “runWolfPsortSummaryplant”, which estimates localization sites with a sensitivity and specificity of approximately 70%. The NLS was predicted in the mature proteins using NLStradamus (http://www.moseslab.csb.utoronto.ca/NLStradamus/). The potential virulence-related proteins were identified by searching the predicted 758 secreted proteins of A. rabiei against the PHI-base with cut off E-values of ≤ 1e-5.The predicted secretome was functionally annotated by assigning GO terms using BLAST2GO. The CAZymes Analysis Toolkit (CAT) was used to detect carbohydrate active enzymes (CAZymes) based on the CAZy database in the Accession numbers. The Ascochyta rabiei whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JYNV00000000. The version described in this paper is the first version, JYNV01000000.How to cite this article: Verma, S. et al. Draft genome sequencing and secretome analysis of fungal phytopathogen Ascochyta rabiei provides insight into the necrotrophic effector repertoire. Sci. Rep. 6, 24638; doi: 10.1038/srep24638 (2016).


Encarsiagenus (Hymenoptera: Aphelinidae) are important biological control agents against whiteflies. Some of the species in this genus not only parasitize their hosts, but also kill them through host feeding. The whitefly parasitoid,Encarsia formosaGahan, was examined to determine whether the rearing host species affects its subsequent host-feeding capacity and parasitism.E. formosawasps were reared onTrialeurodes vaporariorum(Westwood) (Hemiptera: Aleyrodidae) andBemisia tabaci(Gennadius) ‘Q’, and their subsequent host-feeding capacity and parasitism ofT. vaporariorumandB. tabaciwere examined.E. formosareared onT. vaporariorumwere significantly larger in body size than those reared onB. tabaci, but these wasps killed a similar number of whitefly nymphs by host feeding when they attacked the same host species on which they were reared. Regardless of the species on which it was reared,E. formosafed significantly more on theB. tabacinymphs than on theT. vaporariorumnymphs. The number of whitefly nymphs parasitized byE. formosadiffered between the wasps reared onT. vaporariorumand those reared onB. tabacidepending on which whitefly species was offered as a host. In addition, the wasps reared onT. vaporariorumparasitized significantly more onT. vaporariorumthan those reared onB. tabaci. The wasps reared onB. tabaci, however, parasitized similar numbers of whiteflies of both host species. The results indicated that the host-feeding capacity ofE. formosawas affected more by the host species attacked than by the rearing host species, but the parasitism was affected by the host species attacked and the rearing host species. Generally,E. formosareared onT. vaporariorumkilled moreT. vaporariorumnymphs by parasitism and host feeding than those reared onB. tabaci.Additionally, a similar number ofB. tabacinymphs were killed by parasitism and host feeding regardless of the rearing host species. Currently coexistence ofB. tabaciandT. vaporariorumon vegetable crops usually occurs in some areas; our results may provide helpful information on using mass-reared parasitoids against mixed whitefly infestations in biological control programs.Parasitoids of the Some species of insect parasitoids, particularly species of the order Hymenoptera, parasitize or deposit their eggs in their hosts and feed on the hosts (host feeding), which often results in host mortality . Host fEncarsia formosaGahan (Hymenoptera: Aphelinidae), one of the most successfully commercialised natural enemies used for the biological control of insect pests, has been used to control the whitefliesTrialeurodes vaporariorum(Westwood) (Hemiptera: Aleyrodidae) andBemisia tabaci(Gennadius) in many countries throughout the world . This parasitoid is a typical non-concurrent destructive host feeder that uses different host individuals for oviposition and host feeding  in August 2008. Following procurement, the parasitoids were continuously kept in an insectary ) in separate screened cages (60 × 60x60 cm). The biotype ‘Q’ is a cryptic species ofB. tabaci and enclosed in whitefly-proof screened cages. Plants with six fully-extended leaves were used in the experiments.E. formosa(< 3 h) reared onT. vaporariorumorB. tabaciwas introduced onto a detached tomato leaf with 40 third instars ofT. vaporariorumorB. tabaci.The third instars ofT. vaporariorumorB. tabaciwere selected as attacked hosts because they are preferred for parasitism and host feeding byE. formosa(Tomato leaves with nearly emerged parasitoids were placed in large Petri dishes (15.0 cm diam and 1.5 cm deep) and were monitored every 10 min. A newly emergedformosa.B. tabacior 40 adults ofT. vaporariorumwere introduced onto the lower surface of the leaf of a potted tomato plant in a clip cage (4.0 cm diam) for oviposition for 12 h. The nymphs were then monitored daily until they developed into third instars. Forty nymphs of the desired stage were used on each leaf, and extra whitefly nymphs were removed under a binocular stereoscopic microscope using an insect pin. The petiole end of the detached tomato leaf with whitefly nymphs was wrapped with cotton, inserted into a 60-mL cup full of water, and then placed in a clear plastic cup  with a ventilation hole (4 × 4 cm) and covered with a 100-mesh polyethylene screen. This clear plastic cell was used to evaluate the host feeding and parasitism byE. formosaon the whitefly nymphs . The development of the parasitoids was monitored daily until the adults emerged. These adults, reared on eitherB. tabaciorT. vaporariorum(<12 h), were collected in 10-mL glass vials and placed at—20°C for 2 h. The head width, body length, and hind tibia length for each individual parasitoid were measured, as described byE. formosaindividuals each reared onT. vaporariorumandB. tabaci.Three females ofE. formosareared on different host species with respect to their host-feeding capacity and parasitism on different host species. The two factors analysed were the rearing host species (two levels) and the attacked host species (two levels). The means were separated with Tukey HSD test atP< 0.05. A Studentt-test was used to analyze the body size ofE. formosareared onT. vaporariorumandB. tabaci. All of the statistical analyses were performed using the DPS (Data Processing System) software  was performed to compareoftware .E. formosa. However, the host species attacked had a significant effect on host feeding . There was no difference in the number of whitefly nymphs killed by host feeding between theE. formosareared onT. vaporariorumand those reared onB. tabaciwhetherT. vaporariorumorB. tabaciwere offered as hosts . The number of whitefly nymphs killed byE. formosathrough parasitism differed between theE. formosareared onT. vaporariorumand those reared onB. tabacidepending on which whitefly species were offered as hosts. IfT. vaporariorumwas used as the host, the wasps reared onT. vaporariorumparasitized more whiteflies than those reared onB. tabaci. IfB. tabaciwas used as the host, however, the wasps reared onB. tabaciparasitized more whiteflies than those reared onT. vaporariorum. The total number of whiteflies killed by host feeding and parasitism differed between theE. formosareared onT. vaporariorumand those reared onB. tabacidepending on which whitefly species was offered as hosts. IfT. vaporariorumwas used as the host, the wasps reared onT. vaporariorumandB. tabacicaused similar numbers of whitefly mortality through parasitism and host feeding. IfB. tabaciwas used as the host, however, the wasps reared onB. tabacikilled significantly more whiteflies than those reared onT. vaporariorum.TheMetaphycus helvolus(Compere) (Trichogramma turkestanicaMeyer (Encarsia sophia(Girault and Dodd)  ,TrichoaMeyer , andEnd Dodd) , egg lod Dodd) , host sd Dodd) . Earlied Dodd) , 2010 fd Dodd) . In thiTrichogramma,T. pretiosumRiley reared onEphestia kuehniellaZeller parasitized more eggs ofE. kuehniellaorSitotroga cerealella(Olivier) than those reared onS. cerealella(T. minutumreared onChoristoneura fumiferana(Clemens),Lambdina fiscellaria(Guenee), orManduca sexta(L.) attacked significantly more eggs ofE. kuehniellathan those reared onE. kuehniella than on third and fourth instars (larger hosts) ofB. tabaci. For nymphs of the same stage,T. vaporariorumis larger thanB. tabaciin body size, andE. formosareared on eitherT. vaporariorumorB. tabacifed more on the third instars ofB. tabacithan on the same stage nymphs ofT.Hosts vary in quality for oviposition and the host feeding of parasitoids, and host size is the most ubiquitous factor contributing to host quality . As shovaporariorum.E. formosareared onT. vaporariorumorB. tabaciparasitized significantly more hosts of the whitefly species on which they were reared, which may be a result of long-term adaption to the species. Certainly, the host on whichE. formosais reared affects the subsequent host selection either because of a genetic response to selection or as a result of environmental conditioning (tioning .E. formosareared onT. vaporariorumare clearly larger than those reared onB. tabaci. As expected,E. formosareared onT. vaporariorumkilled moreT. vaporariorumnymphs by parasitism than those reared onB. tabaci. Additionally, a similar number ofB. tabacinymphs were killed byE. formosaregardless of the rearing host species. Furthermore,E. formosafed on a similar number ofT. vaporariorumandB. tabacinymphs regardless of the rearing host species. The host-feeding behavior in parasitoids does not appear to be correlated with the rearing host. In general, theE. formosareared onT. vaporariorumexhibit strong biocontrol effectiveness against whiteflies; in this study, they killed moreT. vaporariorumand a similar number ofB. tabacinymphs by parasitism and host feeding than those reared onB. tabaci. With the rapid widespread invasion ofB. tabaci‘B’ and ‘Q’, currently mixed infestations ofB. tabaciandT. vaporariorumon vegetable crops usually occur in some countries (E. formosamass-reared onT. vaporariorumagainst mixed whitefly infestations in biological control programs.The larger parasitoids produced from larger hosts, as compared with those from smaller hosts, have exhibited superiority in fecundity and longevity (Hohmann et al. 1988,untries . Our re


Objective. High-molecular-weight adiponectin (HMW-adiponectin) and leptin are two important adipokines. The aim of this study was to examine the association between the two adipokines and anthropometric measurements of neonates at birth. Furthermore, we would like to explore whether HMW-adiponectin and leptin correlate with insulin sensitivity in neonates. Methods. Venous cord blood samples were obtained from 266 full-term healthy neonates consecutively born at Peking Union Medical College Hospital. HMW-adiponectin, leptin, blood glucose, and insulin concentrations were measured. Results. HMW-adiponectin and leptin were significantly higher in females compared with males . Univariate correlation analysis showed that leptin concentrations in cord blood were positively associated with gestational age, birth weight, body length, ponderal index, placenta weight, insulin, and insulin sensitivity . However, there was no correlation between cord blood HMW-adiponectin levels and foetal anthropometric measurements or foetal insulin sensitivity indicators . Multivariate linear regression analysis indicated that leptin  in cord blood was independently associated with insulin sensitivity. Conclusions. Leptin concentrations, but not HMW-adiponectin, were positively associated with foetal anthropometric measurements. Leptin concentrations are significantly associated with foetal insulin sensitivity, and there were no significant correlations between HMW-adiponectin levels and foetal insulin sensitivity. Currently, diabetes mellitus poses a major challenge to public health. This issue is particularly important in light of the increasing prevalence of obesity, insulin resistance, and type 2 diabetes mellitus among children and adolescents . Currentβ cells. Some clinical studies have shown that serum leptin concentrations are directly proportional to fat mass, and decreased central leptin responsiveness has been observed in obese patients [Leptin is regarded as a prototypical adipokine. It can control body weight and regulate energy balance mainly through neurons in the arcuate nucleus of the hypothalamus. Furthermore, it can suppress insulin secretion from pancreatic patients . Leptin patients . HoweverAdiponectin has been widely studied because of its diverse biological functions, including anti-inflammatory, antiatherogenic, antidiabetic, and cardioprotective effects . It circThe majority of relevant studies indicated that adiponectin and leptin levels in cord blood were associated with anthropometric measurements at birth –14. MoreInformed parental consent was obtained from each participant, and the study protocol was approved by the Institutional Review Board and Ethics Committee of Peking Union Medical College Hospital (PUMCH) and the Ethics Committee of PUMCH (S-002).The study population consisted of 266 full-term neonates  consecutively born at Peking Union Medical College Hospital from July 2009 to January 2011. Infants whose mothers had multiple-fetus pregnancy, chronic hypertension, pregnancy-induced hypertension, gestational diabetes mellitus, endocrine disorders, and other severe maternal illnesses, and infants with a gestational age <37 or >42 weeks, asphyxia at birth, intrauterine infection, and congenital anomalies, were excluded from the study.3). The body weight of each neonate and placental weight were determined to the nearest 1 g using an electronic scale. Body length was determined to the nearest 0.1 cm in the supine position with a length board. Head circumference was determined with a plastic tape to the nearest 0.1 cm.Data and specimens were collected at delivery. Three trained research nurses and assistants collected data on maternal, pregnancy, and birth characteristics using structured study questionnaires through face-to-face interviews and medical chart reviews. The variables of the neonates included birth weight, length, head circumference, gestational age (GA), and ponderal index (PI). The clinical characteristics and anthropometric measurements are shown in μU/mL).Venous cord blood samples were obtained from 266 newborns immediately after birth with EDTA as anticoagulant. All plasmas were obtained by centrifugation at 4000 ×g for 10 min in a microcentrifuge at room temperature and stored in multiple aliquots at −80°C until biochemical assays. Plasma glucose  was measured by an automated glucose oxidase method. Insulin was measured by a Human Insulin-Specific RIA radioimmunoassay (RIA) kit . The inter- and intra-assay coefficients of variation were 2.9–6.0% and 2.2–4.4%, respectively. Leptin was measured by a human leptin RIA kit . The inter- and intra-assay coefficients of variation were 3.6–6.2% and 3.4–8.3%, respectively. The plasma HMW-adiponectin concentration was determined by a quantitative ELISA (enzyme-linked immunosorbent assay) kit . The sensitivity was 0.989 ng/mL. The intra- and interassay coefficients of variation were 2.6–3.7% and 8.3–8.6%, respectively. All samples were tested in duplicate in a blinded manner. Although the euglycaemic insulin clamp technique is the best available standard for measurement of insulin action, it was impossible to apply at birth. The cord plasma G/I (glucose/insulin ratio), which is a simple estimate of insulin sensitivity using insulin and glucose levels , 17, wast-test or the Mann-Whitney U test. Distributions were tested for normality using the Shapiro-Wilk W test. Log transformations were applied for variables with skewed data distribution in all comparisons before correlation and regression analyses. Pearson correlation analysis was used to assess the relationships between HMW-adiponectin and some anthropometric measurements and biochemical characteristics. Multiple regression analysis assessed whether the relationship between leptin and insulin sensitivity is independent of the other clinical parameters. Two-tailed P values <0.05 were considered statistically significant. All statistical analyses were calculated with SPSS 15.0 .The data were expressed as the mean ± standard deviation (SD). We compared baseline characteristics between male and female groups using an unpaired P = 0.02), body length , and birth weight to length ratio  were significantly higher in male than female subjects. However, females had higher HMW-adiponectin and leptin concentrations compared with males . No significant differences were detected in insulin sensitivity between male and female neonates.HMW-adiponectin is present abundantly in cord blood. Cord blood concentrations of HMW-adiponectin, glucose, insulin, and G/I are shown in r = 0.24, P = 0.001), birth weight , body length , ponderal index , and placenta weight . Furthermore, leptin concentrations were positively associated with insulin  and negatively associated with G/I . However, there was no correlation between cord blood HMW-adiponectin levels and foetal anthropometric measurements . HMW-adiponectin concentrations in cord blood were also positively associated with insulin  and leptin . However, no correlation was found between cord blood HMW-adiponectin levels and foetal insulin sensitivity indicator (P > 0.05)  .B = −0.126, P = 0.045) was independently associated with insulin sensitivity as estimated by G/I after adjusting for HMW-adiponectin, gestational age, birth weight, body length, head circumference, placenta weight, and the type of delivery (Having demonstrated that leptin in cord blood is positively associated with insulin sensitivity in the foetus at birth, we next queried whether the relationship between leptin and insulin sensitivity is independent of other clinical parameters. Multivariate stepwise regression analysis with logarithmically transferred G/I as a dependent variable was applied to investigate the relationship. Leptin (delivery .To our knowledge, so far, our present study has relatively more clinical foetal samples compared with any other study, which could indicate a more convincing result , 18–20. In our present study, the results indicated that HMW-adiponectin was significantly higher in females compared with males. Similar results were shown in another study . A recenThe results of our present study also indicated that leptin levels in cord blood are negatively correlated with foetal insulin sensitivity. Consistently, two other studies focused on the foetal leptin and insulin sensitivity, which also demonstrated a negative correlation , 30. TheOur findings suggest that leptin, but not HMW-adiponectin, is a useful biomarker for the evaluation of the metabolic status of neonates, distinct from that of adults. Adipokines may play a critical role in the growth, development, and glucose metabolism of the neonates.


H. pylori is the Vacuolating cytotoxin A (VacA) named after its ability to induce the formation of “vacuole”-like membrane vesicles in the cytoplasm of gastric cells. VacA has been associated with the disruption of mitochondrial functions, stimulation of apoptosis, blockade of T cell proliferation and promotion of regulatory T cells, thereby making it a promising vaccine target. Immunity to bacterial virulence factors is well known to protect humans against bacterial infections; hence, detoxified VacA has been evaluated as a vaccine antigen. Our short review summarizes the pre-clinical and clinical data that have been published on the use of VacA in the development of the H. pylori vaccine.One of the major toxins secreted by  Helicobacter pylori infection is one of the most common infections in human beings worldwide [H. pylori colonizes the mucus gastric layer [H. pylori organisms are free living in the mucus layer, but some organisms attach to gastric epithelial cells [orldwide . After eic layer  but doesic layer , and theal cells  and smalal cells .H. pylori uses urease and α-carbonic anhydrase to generate ammonia and HCO32− enabling it to colonize and function at low pH conditions [H. pylori penetrates the mucus layer [+/K+ ATPase [To survive into the stomach, nditions ,6. Furthus layer . Once es+ ATPase ,9.H. pylori virulence factors, environmental factors and the host immunological status to bacterial infection, H. pylori infection can lead to several clinical complications such as gastritis, peptic ulcer disease, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma [H. pylori [H. pylori strains [H. pylori, have been evaluated [Depending on lymphoma ,11,12. H. pylori ,14. Howe strains ,14. Thervaluated .H. felis bacterial lysates or chemically inactivated whole-cell bacteria delivered to mice orogastrically along with cholera toxin (CT) or E. coli heat-labile enterotoxin (LT) as mucosal adjuvants [The first murine vaccine studies utilized djuvants . HoweverBordetella pertussis infections [H. pylori vaccine [H. pylori has a large arsenal of virulence factors that help the bacteria to colonize and persist within the stomach mucosa of its host. One of these virulence factors, Urease is essential for colonization and pathogenic events and due to its abundance has made it an ideal vaccine candidate [H. felis or H. pylori infections in mice and ferrets [H. pylori Urease B subunit fused to LT B subunit significantly reduced the acquisition of natural H. pylori infection in children [H. pylori infection, more work needs to be done in order to enhance its protective capacity. Indeed, whereas the vaccine protected more than 70% of recipients 1-year post vaccination, only 56% were protected 2–3 post vaccination [H. pylori vaccine containing other virulence factors may be considered to enhance vaccine efficacy. VacA is one of the virulence factors that have been evaluated as vaccine antigen.Immunity to bacterial virulence factors is known to protect humans against bacterial infections. A well-known example is the use of detoxified pertussis toxin, filamentus hemagglutin and pertactin in the acellular pertussis vaccine to protect against fections . A simil vaccine . Indeed,andidate . In addiandidate . Differe ferrets . Althoug ferrets , Zeng anchildren . While tcination ,21. TherH. pylori to establish a chronic infection [H. pylori derived virulence factors, including NAP (Neutrophil-activating protein), and CagA constitute a complex network to regulate chronic gastric injury and inflammation that lead to gastric carcinogenesis [VacA is a secreted protein that has been reported to induce vacuolization, membrane anion-selective channel and pore formation, disruption of endosomal and lysosomal activity, and apoptosis in target cells. Furthermore, it has also been shown to induce IL-8 production in monocytes derived cell line . Finallynfection ,24. VacAogenesis .H. pylori express a form of VacA. However, polymorphism may affect VacA toxigenic and pathogenic activity [vacA are important for VacA toxicity. The vacA signal region encodes the N terminus of the toxin and may be active (type s1) or encodes an N-terminal extension, which blocks activity (s2) [vacA mid region may bind to a wide range of cells, causing toxicity (type m1), or to a smaller range (type m2) of cells [Nearly all strains of activity ,26,27. Iof cells . Importaof cells . They shof cells ,30.Escherichia coli and purified by affinity chromatography is nontoxic [H. pylori, the E. coli-expressed recombinant VacA was inactive in a HeLa cell assay of vacuolization and failed at inducing high titers of neutralizing antibodies in rabbits. Hence, the neutralizing epitopes in the native toxin protein are likely to be conformational and may exist only when the protein folds correctly into its native structure [VacA cytotoxicity causes acute gastric epithelial erosion and ulceration, which excludes the use of the active toxin in humans . VacA tonontoxic  and is atructure .H. pylori infection but also prevents a subsequent reinfection in the majority of vaccinated animals [H. pylori challenge [et al. The authors fused a fragment of VacA (aa 744–805) that contains CD4+ T cell epitopes to CagA and urease B fragments and expressed the fusion protein into an attenuated Salmonella vector [Salmonella expressing the CagA-VacA-UreB fusion protein, significantly decreased H. pylori colonization [Significant levels of protection were achieved when infected mice were therapeutically vaccinated intragastrically with the non-toxic recombinant VacA combined with the non-toxic mutant of LT, LTK63. Importantly, therapeutic vaccination not only successfully eradicates  animals . The res animals . Althoug animals , the whohallenge . An intea vector . Oral thnization .H. pylori-infected individuals [+ lymphocytes isolated from the gastric epithelium of H. pylori-infected individuals proliferate in an antigen-dependent manner in the presence of VacA [H. pylori infection. However, the detected immune responses directed toward VacA in chronically infected people demonstrate that VacA is immunogenic in the human population and might be a good candidate as vaccine antigen.Anti-VacA antibodies are present in serum and gastric juice from the majority of ividuals  and neutividuals . In addi of VacA ,40. AlthH. pylori strains that produced vacuolating toxin activity in vitro. However, it failed to protect against challenge with a wild-type Tox− strain that lacked detectable toxic activity for HeLa cells [H. pylori strains that lack toxic activity (Tox−) for HeLa cells contain type s1-m2 or s2-m2 vacA alleles [− strain may be due to antigenic diversity among different VacA proteins. In particular, it is possible that T epitopes present in the type s1-m1 VacA fail to cross-react with those present in the type s2-m2 VacA. Therefore, if VacA is to be used as a vaccine antigen, it might be appropriate to immunize with pooled VacA antigens derived from several different VacA families and/or include additional virulence factors of H. pylori [Immunization with purified type s1-m1 VacA conferred protection against subsequent challenge with two La cells ,41. It i alleles ,35,41. B. pylori .et al. evaluated a multicomponent vaccine composed of purified recombinant VacA, CagA and NAP in a human phase I clinical trial [H. pylori [H. pylori-negative human volunteers [In vitro, VacA restimulation of peripheral blood mononuclear cells induced cellular proliferation, which was paralleled by production of IFN-γ, suggesting that the vaccine had induced antigen-specific T-cell responses. Remarkably, a booster immunization 18–24 months after primary immunization elicited a strong anamnestic antibody response to VacA in all subjects, and VacA specific production of IFN-γ can be detected after in vitro VacA stimulation of peripheral blood mononuclear cells. These results suggested that the vaccine induced long-lasting immunity and immunologic memory in the B and T cell populations [Malfertheiner P al trial . Previou. pylori  and erad. pylori . The vaclunteers . The vaculations . AlthougHelicobacter specific CD4+ T cell response associated with an inflammatory reaction to reduce Helicobacter infection [H. pylori [+ T cells will secrete a large set of cytokines such as IFN-γ, IL-17 and TNFα that promote the recruitment and the activation of inflammatory cells such as neutrophils [H. pylori in mice [H. pylori vaccine, these pre-clinical data suggest that it might be of high interest to correlate the presence of circulating and/or stomach VacA-specific memory CD4+ T cells with the vaccine efficacy: hence, providing an immune correlate of vaccine protection.The demonstration that VacA injected to healthy individuals induces an anamnestic T cells response is of particular interest . Indeed,nfection . Since, . pylori ,40 or in. pylori , it can trophils , inflammtrophils  or mast trophils  which pa in mice . In respH. pylori isolates, limiting its potential as vaccine candidate if used alone, pre-clinical and clinical data clearly demonstrate the good potential of the molecule as vaccine antigen. However, since VacA is a protein that modulates many biological processes of gastric epithelial cells and immune cells [Although heterogeneity exists in the expression of VacA in ne cells ,24,25, a


Plasmodium and is one of the most prevalent infectious diseases in tropical and subtropical regions. For this reason, effective and practical diagnostic methods are urgently needed to control the spread of malaria. The aim of the current study was to identify a panel of new malarial markers, which could be used to diagnose patients infected with various Plasmodium species, including P. knowlesi, P. vivax and P. falciparum. Sera from malaria-infected patients were pooled and compared to control sera obtained from healthy individuals using the isobaric tags for relative and absolute quantitation (iTRAQ) technique. Mass spectrometry was used to identify serum proteins and quantify their relative abundance. We found that the levels of several proteins were increased in pooled serum from infected patients, including cell adhesion molecule-4 and C-reactive protein. In contrast, the serum concentration of haptoglobin was reduced in malaria-infected individuals, which we verified by western blot assay. Therefore, these proteins might represent infectious markers of malaria, which could be used to develop novel diagnostic tools for detecting P. knowlesi, P. vivax and P. falciparum. However, these potential malarial markers will need to be validated in a larger population of infected individuals.Malaria is caused by parasitic protozoans of the genus  Plasmodium. Among the Plasmodium species, P. falciparum, P. vivax, P. knowlesi, P. malariae and P. ovale are known to infect humans under natural conditions [Plasmodium species are known as the human malaria species. However, P. malariae and P. ovale are rare and less dangerous compared to other species [Malaria has been reported to affect over two billion people globally, impacting numerous countries. In fact, according to the World Health Organization’s World Malaria Report 2013 and the Global Malaria Action Plan, there are currently 3.4 billion people at risk of malaria infection in 97 countries and territories [Plasmodium species display distinct characteristics. P. knowlesi primarily causes chronic infection in long-tailed and pig-tailed macaques, resulting in several life-threatening complications, including renal failure, liver failure, and several non-malarial symptoms. However, in terms of hematological analysis, the clinical manifestation of knowlesi malaria in humans involves hypoglycemia, anemia, and hyperbilirubinemia [P. falciparum causes a diffuse encephalopathy called cerebral malaria (CM), which is the principal cause of malaria-related death. Notably, angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2), which are major regulators of angiogenesis, have been used to identify CM severity [P. vivax, which displays a unique life cycle, is one of the oldest-known parasites infecting humans [P. vivax are delivered to humans via spz-infected mosquitoes and invade human hepatocytes. Subsequently, the spz either develop into merozoites within infected hepatocytes or remain in a dormant stage as hypnozoites. Activation of dormant hypnozoites following a primary attack can result in an additional blood stage called a relapse [There are various perceptions regarding the source of malaria. While midwives and pregnant women consider mosquitoes to be the main transmitters of malaria in East Sudan , Nigeriaubinemia . P. falcseverity . While Aseverity . Thus, cseverity . P. vivag humans . In the  relapse .The ability to accurately measure and compare protein expression levels is one of the most important goals in post-genomics malaria research. Earlier studies have utilized two-dimensional gel electrophoresis to analyze malarial proteins . In addiP. knowlesi (n = 9), P. vivax (n = 6) or P. falciparum (n = 10). In addition, 23 samples were obtained randomly from normal healthy individuals. It is known that the identification of potential serum biomarkers can be complicated by the high abundance of proteins in serum samples [At the UMMC, serum samples were collected from 25 newly diagnosed malaria patients infected with  samples . Thus, tP. knowlesi, P.falciparum and P.vivax) relative to uninfected controls.A total of 152 proteins (≥95% confidence) were detected following albumin depletion . Two uprDownregulated proteins generally serve as effective biomarkers. Therefore, Western Blotting was used to further validate our findings regarding decreased of HAP expression. Upon Western Blotting analysis, HAP was solely present in control sera, but was lacking in three malaria-infected groups . These fP. vivax, P. falciparum, or P. knowlesi were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) nitrocellulose membrane. Membranes were subsequently immunoblotted with monoclonal antibody against HAP.Unfractionated serum samples from patients infected with P. knowlesi, P.falciparum or P. vivax) and non-infected healthy subjects. The present data has validated the expression of CADM4 and HAP in other studies [P. knowlesi, P. falciparum and P. vivax.In the present study, iTRAQ has been used as a quantitative proteomic technique to analyze sera from patients infected with malaria , P. vivax (n = 6) or P. falciparum (n = 10)) at the University of Malaya Medical Centre . In order to confirm the Plasmodium species infecting each patient, samples were screened using conventional light microscopy, P. falciparum histidine-rich protein-2 (PfHRP2)-based lateral flow rapid diagnostic kits, and nested polymerase chain reaction (PCR) [n = 23) were obtained randomly from normal healthy individuals. All subjects gave their consent for participation, and the study was approved by the Ethical Committee of UMMC in accordance with the International Conference on Harmonization-Good Clinical Practice (ICH-GCP) guidelines and the declaration of Helsinki.Blood was collected from newly diagnosed malaria patients (on (PCR) . Also, cvia centrifugation . After the sample was concentrated to less than 25 μL, 300 μL of ASM Buffer 2 (provided in the kit) was added to the spin column. The sample was again concentrated to a volume less than 25 μL. The ToPA™ assay kit  was used to determine protein concentrations.Albumin was depleted using the Albumin Segregation Kit  according to the manufacturer’s protocol. Briefly, albumin segregation matrix (ASM), contained within a macrospin column, was washed three times with ASM Buffer 1 (included in the kit). The buffer was removed by centrifugation . Serum sample was diluted with ASM Buffer 1 to a final volume of 300 μL and was mixed by gentle vortexing. The diluted serum was then transferred to the ASM-containing macrospin column. Following incubation (1 min at room temperature), the column was centrifuged  without drying the matrix. The flowthrough was again added to the ASM column and was centrifuged. This process was repeated once more, and the final flowthrough was saved. The ASM column was washed with ASM Buffer 1 (300 μL) and centrifuged . The flowthrough from this washing step was combined with the previously saved flowthrough. This combined sample was finally concentrated with a 5-kDa molecular weight cutoff (MWCO) spin column P. vivax, P. falciparum and P. knowlesi (27 μg each) samples were used in the iTRAQ analysis. Sample preparation and labeling for iTRAQ were performed based on the manufacture’s protocol . One microliter of 2% sodium dodecyl sulfate (SDS) was added to denature the proteins (mixed by vortexing). Disulfide groups were reduced by incubation at 60 °C for 1 h with 50 mM Tris-(2-carboxyethyl)-phosphine. The samples were then cooled, and the free cysteine groups were blocked with 200 mM methyl methanethiosulfonate in isopropanol (incubated for 10 min at room temperature). Trypsin was then added to the samples , which were incubated overnight at 37 °C. Following trypsin digestion, control, P. vivax, P. falciparum, and P. knowlesi samples were labeled with isobaric tags for 2 h . After labeling, the digested and labeled samples were combined and vortexed. The final mixed sample was cleaned using an SCX column to remove iTRAQ reagents, reducing agents, alkylating agents, and other impurities. The peptides were eluted from the column using 450 mM ammonium acetate buffer (pH 2.0). The eluted peptides were dried using a speedvac to remove the ammonium acetate. Dried peptides were dissolved in 0.1% formic acid containing 5% acetonitrile, and 15 μL of this solution was injected into the LTQ XL™ mass spectrometer .Serum proteins from control, m/z: 400–1500) followed by MS/MS scans on the three most abundant ions. Each MS/MS analysis was followed by pulsed Q dissociation (PQD) activation for iTRAQ reporter ion fragmentation, allowing for peptide quantitation.All LC-MS/MS analyses were performed using the ThermoElectron ProteomeX Workstation , which includes a high-performance liquid chromatography HPLC front end and an LTQ XL™ nano-spray-ion-trap mass spectrometer back end. The dried, trypsin-digested peptides were re-suspended in 20 μL of 0.1% formic acid containing 5% acetonitrile. Subsequently, 15 μL of the mixture was loaded onto a Thermo PicoFrit C18 nanospray column and analyzed by HPLC. The peptides were eluted from the column using a linear acetonitrile gradient (2%–45% over 230 min) into the LTQ XL™ mass spectrometer via a nanospray source . A data-dependent Top 3 method was used for peptide identification, involving a full MS scan . Search parameters were set for trypsin digestion, allowing for up to two missed cleavages per peptide. In addition, static modification C 57.0215, N-term 144, and K 144 were set as fixed modifications. Oxidation of methionine was used as variable modification. Peptides were filtered subject to the default charge Sera from the control individuals and each of the three malaria patients were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12.5% gel. Western blotting was performed following electrophoretic transfer of the proteins onto polyvinylidene difluoride (PVDF) membranes as previously described ,14. MembP. vivax, P. falciparum or P. knowlesi infection. However, further validation of these findings will need to be carried out in more extensive clinically representative populations.In summary, the data of this study suggest that HAP, CRP, and CADM4 could be used as panel biomarkers of