The Rickettsia prowazekii invasion gene homolog (invA) encodes a Nudix hydrolase active on adenosine (5')-pentaphospho-(5')-adenosine

The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (Np(n)N; n > or = 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5')-pentaphospho-(5')-adenosine into ATP and ADP with a K(m) of 0.1 mM and k(cat) of 1.9 s(-1). Guanosine (5')-pentaphospho-(5')-guanosine and adenosine-(5')-hexaphospho (5')-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg(2+) or Zn(2+), for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion.     

The mammalian cell entry (Mce) protein of pathogenic Leptospira species is responsible for RGD motif-dependent infection of cells and animals

Mammalian cell entry proteins (Mces) contribute to Mycobacterium tuberculosis virulence. A mce homologue has been identified in the Leptospira interrogans genome, but its function was unknown. We showed that the mce gene is expressed only by pathogenic Leptospira strains tested. Leptospiral mce mRNA and Mce protein levels increased during infection of macrophages. The ability to infect macrophages was significantly lower in a strain with the mce gene deleted (mce^‐). Complementation of the mce gene restored the ability of the mutant strain (mce^com) to adhere to and invade cells. Importantly, the mce gene knock‐in strain (mce⁺) derived from L. biflexa acquired the ability to infect cells, and the mce^+/ΔRAA knock‐in strain (in which the RGD motif was replaced by RAA) was unable to infect cells. The mce^‐ mutant was also dramatically less efficient in infecting hamsters than the wild‐type L. interrogans strain, and fewer leptospires of the mutant were found in peripheral blood monocytes and the urine from infected animals. The recombinant Mce protein showed a high binding affinity to the integrins α5β1 and α_Vβ3. Blockade of the two integrins or the Mce protein decreased leptospiral adherence and invasiveness. The results showed that Mce is an RGD‐motif‐dependent virulence factor in pathogenic Leptospira species.     

Comparative proteomic analysis of differentially expressed proteins in the urine of reservoir hosts of leptospirosis

Rattus norvegicus is a natural reservoir host for pathogenic species of Leptospira. Experimentally infected rats remain clinically normal, yet persistently excrete large numbers of leptospires from colonized renal tubules via urine, despite a specific host immune response. Whilst persistent renal colonization and shedding is facilitated in part by differential antigen expression by leptospires to evade host immune responses, there is limited understanding of kidney and urinary proteins expressed by the host that facilitates such biological equilibrium. Urine pellets were collected from experimentally infected rats shedding leptospires and compared to urine from non-infected controls spiked with in vitro cultivated leptospires for analysis by 2-D DIGE. Differentially expressed host proteins include membrane metallo endopeptidase, napsin A aspartic peptidase, vacuolar H+ATPase, kidney aminopeptidase and immunoglobulin G and A. Loa22, a virulence factor of Leptospira, as well as the GroEL, were increased in leptospires excreted in urine compared to in vitro cultivated leptospires. Urinary IgG from infected rats was specific for leptospires. Results confirm differential protein expression by both host and pathogen during chronic disease and include markers of kidney function and immunoglobulin which are potential biomarkers of infection.     

Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family.

One of the earliest steps in the pathogenic cycle of the facultative intracellular pathogen Salmonella spp. is the invasion of the cells of the intestinal epithelium. We have previously identified a genetic locus, inv, that allows Salmonella spp. to enter cultured epithelial cells. invA is a member of this locus, and it is the first gene of an operon consisting of at least two additional invasion genes. We have constructed strains carrying nonpolar mutations in invA and examined the individual contribution of this gene to the invasion phenotype of Salmonella typhimurium. Nonpolar S. typhimurium invA mutants were deficient in invasion of cultured epithelial cells although they were fully capable of attaching to the same cells. In addition, unlike wild-type S. typhimurium, invA mutants did not alter the normal architecture of the microvilli of polarized epithelial cells nor did they cause any alterations in the distribution of actin microfilaments of infected cells. The invasion phenotype of invA mutants was readily rescued by wild-type S. typhimurium when cultured epithelial cells were simultaneously infected with both strains. On the contrary, in a similar experiment, the adherent Escherichia coli strain RDEC-1 was not internalized into cultured cells when coinfected with wild-type S. typhimurium. The invA locus was found to be located at about 59 min on the Salmonella chromosome, 7% linked to mutS. The nucleotide sequence of invA showed an open reading frame capable of encoding a polypeptide of 686 amino acids with eight possible membrane-spanning regions and a predicted molecular weight of 75,974. A protein of this size was visualized when invA was expressed in a bacteriophage T7 RNA polymerase-based expression system. The predicted sequence of InvA was found to be homologous to Caulobacter crescentus FlbF, Yersinia LcrD, Shigella flexneri VirH, and E. coli FlhA proteins. These proteins may form part of a family of proteins with a common function, quite possibly the translocation of specific proteins across the bacterial cell membrane.     

Development of species-specific PCR primer sets for the detection of Leptospira

Spirochetes of the genus Leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis. Rapid and simple assays for the identification of individual Leptospira species are currently not available. For identification of individual Leptospira species, PCR primers that detect the ompL1 gene sequence for the majority of pathogenic leptospires were developed in this study. The primer pairs detect Leptospira interrogans, Leptospira borgpetersenii, Leptospira kirschneri, Leptospira santarosai, Leptospira weilii and Leptospira noguchii, without cross-reacting with other Leptospira species. The development of the primers revealed a divergence of the ompL1 gene within L. interrogans, splitting this species into two separate groups. The species-specific primers will be especially useful in epidemiological studies and disease outbreak investigations for the detection of Leptospira species in human, animal and environmental samples.     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

The OmpA-like protein Loa22 is essential for leptospiral virulence

Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has a predicted OmpA domain based on sequence identity. The resulting mutant did not express Loa22 and was attenuated in virulence in the guinea pig and hamster models of leptospirosis, whereas the genetically complemented strain was restored in Loa22 expression and virulence. Our results show that Loa22 was expressed during host infection and exposed on the cell surface. Loa22 is therefore necessary for virulence of L. interrogans in the animal model and represents, to our knowledge, the first genetically defined virulence factor in Leptospira species.     

The usefulness of semi‐solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka,Japan

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi‐solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira‐positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi‐solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi‐solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.     

Unique cell adhesion and invasion properties of Yersinia enterocolitica O:3, the most frequent cause of human Yersiniosis

Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment.     

Survival rate of Leptospira pomona in the soil at a natural leptospirosis focus

At the area of a natural focus of leptospirosis (caused by L. pomona) in the Mozdok region of the North Occetic ASSR leptospires were detected by the method of dark field microscopy in 22% of intact soil samples. The presence of pathogenic leptospires in this soil was not confirmed by the method of the biological assay on Syrian golden hamsters. In controlled tests lasting 1-277 days, in 30.4% of cases L. pomona retained their viability, pathogenic and antigenic properties for as long as 74 days, while staying in the soil at the focus of infection with humidity being 15.2-31.4% and pH = 6.7-7.2. 11 Leptospira cultures isolated after staying in the soil retained their pathogenic properties and the death of the animals used in the bioassay from the acute form of leptospiral infection.     

猪链球菌2型Dbp缺失株的构建及特性分析

【目的】通过构建DNA结合膜蛋白(DNA-binding membrance protein,Dbp)基因的缺失株,探究Dbp基因对猪链球菌2型强毒株毒力的影响。【方法】通过PCR检测Dbp基因的分布。利用同源重组原理构建Dbp基因上下游片段的重组质粒,将构建好的质粒电转入ZY05719感受态细胞中,筛选Dbp缺失突变株,通过PCR及测序分析对其进行验证。生物学特性分析比较缺失株?Dbp和野毒株ZY05719在生长速率、形态特征、毒力等方面的差异。【结果】Dbp基因为猪链球菌2型强毒株中相对保守基因,构建了Dbp基因缺失株?Dbp。体外实验结果显示Dbp基因缺失株的生长速率在对数期减慢,并且缺失株?Dbp的荚膜同野毒株存在显著差异,斑马鱼实验结果表明缺失株?Dbp毒力下降。【结论】Dbp与猪链球菌2型的毒力相关,在猪链球菌2型的致病过程中起一定的作用,这丰富了对该菌致病机理的认识。     

Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.     

Crystal structure of the C‐terminal domain of the Salmonella type III secretion system export apparatus protein InvA

InvA is a prominent inner‐membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N‐terminal integral membrane domain and a C‐terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C‐terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V‐type ATPase and a ring building motif found in other T3SS proteins respectively.     

Salmonella typhimurium InvA expression probed with a monoclonal antibody to the C-terminal peptide of InvA

Abstract The Salmonella typhimurium InvA protein is a component of a sec -independent secretion apparatus necessary for full virulence of the bacteria. We generated a monoclonal antibody to the C-terminal portion of the InvA protein that recognized proteins in S. typhimurium and weakly in Y. enterocolitica , but not in several other species of bacteria, including S. flexneri. S. typhimurium grown without agitation produced relatively constant amounts of membrane InvA throughout the growth cycle, whereas bacteria grown with agitation had a sharp increase in the amount of membrane InvA at late exponential phase. Levels of InvA present in Salmonella membranes under some growth conditions do not appear to correlate with levels of invasion under the same conditions.     

问号钩体粘附侵袭相关基因特征分析

使用NCBI ,Swissprot/TrEMBL ,ProDom ,Pfam ,Tmpred ,SignalP ,ClustW等网络资源和软件 ,根据问号钩体黄疸出血型赖株粘附侵袭相关基因诠释结果 ,对mce ,invA ,mviN和atsE四个粘附侵袭相关基因编码蛋白的结构域、跨膜区域和信号肽等进行了详细分析 ,并使用Bioedit,Mega2软件进行氨基酸多重序列比较并绘制系统发生树。结果显示 ,mce和mviN为穿膜蛋白 ,invA和atsE为菌体内蛋白质 ;许多对哺乳动物和对植物致病的微生物具有mce ,invA ,mviN和atsE四个粘附侵袭相关基因 ,其表达的蛋白质在感染宿主过程中起重要作用 ,钩体的粘附侵袭相关蛋白与它们在一级结构上有较高相似性。据生物信息学结果推测 ,问号钩体黄疸出血型赖株粘附侵袭相关基因和钩体致病性间有密切关系 ,其编码蛋白在致病过程中可能起重要作用     

Omp52 is a growth-phase-regulated outer membrane protein of Leptospira santarosai serovar Shermani

We report the expression and characterization of the omp52 gene of Leptospira santarosai serovar Shermani strain CCF that is isolated in Taiwan. omp52 was identified among pathogenic leptospires but not among non-pathogenic leptospires by using suppression subtractive hybridization in our previous study. With an open reading frame of 1371 bp that encodes 456 amino acids and a predicted molecular mass of 52.6 kDa, Omp52 was shown to be an outer membrane protein containing a C-terminal OmpA consensus domain and exposed on the cell surface. Furthermore, Omp52 increases dramatically during the stationary phase, indicating that the expression of Omp52 is environmentally regulated. By using immunoblotting analysis, we proved that Omp52 was expressed in human patients infected with leptospires. These observations suggest that Omp52 may play roles in the interaction of host cells and pathogens during infection.     

Natural Antibody in Mammalian Serum Reacting with an Antigen in Some Leptospires

Serum from normal mammals agglutinated and immobilized nonpathogenic Leptospira biflexa and agglutinated avirulent lines of pathogenic serotypes L. icterohaemorrhagiae and L. zanoni. Virulent lines of L. icterohaemorrhagiae and L. zanoni were not affected, nor were any of three strains of L. pomona, one of which was avirulent. The active principle in serum was a beta-macroglobulin which was heat-labile and reduced by 2-mercaptoethanol, and acted in conjunction with complement and lysozyme; it was absorbable from serum by Formalin-treated susceptible leptospires. The Formalin-stable receptor antigen, named "Z antigen," is associated with virulence rather than pathogenicity, but may not be a determinant of virulence.