Caracterização funcional de proteínas hipotéticas de Leptospira interrogans como adesinas e potenciais antígenos vacinais e para diagnóstico / Functional characterization of hypothetical proteins in Leptospira interrogans as adhesins and potential vaccine and diagnostic antigens

Leptospira spp. constitui um grupo de bactérias espiroquetas gram-negativas englobando espécies saprofíticas, intermediárias e patogênicas, sendo as últimas agentes causadores da leptospirose, doença zoonótica de alcance mundial e endêmica em regiões tropicais em desenvolvimento. O crescente número de espécies identificadas de leptospiras destaca ainda mais sua diversidade genética e mecanismos de virulência únicos, muitos deles com função ainda desconhecida. Esforços para o desenvolvimento de novas vacinas com proteção cruzada e efeito duradouro revelaram possíveis candidatos vacinais que necessitam ser adequadamente validados, sendo assim, há ainda uma urgente necessidade de uma vacina universal contra a leptospirose capaz de controlar e reduzir os surtos cada vez mais frequentes da doença. Adesinas são importantes fatores de virulência em diversos patógenos, constituindo antígenos promissores para o desenvolvimento de vacinas contra a leptospirose, assim como para o desenvolvimento de métodos diagnósticos mais rápidos e precisos. Previamente, foram identificadas três proteínas hipotéticas conservadas em L. interrogans pela técnica de phage display, denominadas arbitrariamente como LepA069, LepA962 e LepA388. A expressão do gene codificador da proteína LepA069 apresentou aumento de aproximadamente 70 % em animais infectados por leptospiras virulentas, representando a primeira evidência funcional desta proteína ainda desconhecida. Porções recombinantes da lipoproteína hipotética LepA962 (LepA962_Nt e LepA962_Phg) foram obtidos, sendo demonstrada a forte interação da proteína LepA962_Phg, contendo a sequência identificada por phage display, com laminina, fibronectina plasmática, colágeno I e fibrinogênio de maneira dose-dependente. Adicionalmente, LepA962_Phg apresentou ligação às células VERO e à sua matriz extracelular secretada, e o soro obtido a partir desta proteína recombinante foi capaz de se ligar à superfície de leptospiras virulentas, indicando que LepA962_Phg pode representar um importante domínio de interação entre as leptospiras e seu hospedeiro. Finalmente, a proteína LepA388 pertencente a uma extensa família de proteínas modificadoras de virulência com função desconhecida (DUF_61), presente apenas nas leptospiras patogênicas mais virulentas, apresentou aumento na expressão de seu gene codificador em animais infectados por leptospiras virulentas de acordo com dados na literatura. Além disso, porções recombinantes da região Nterminal desta proteína apresentaram ligação a laminina, colágenos I e IV, vitronectina e fibronectinas plasmática e celular, principalmente considerando a sequência identificada por phage display. Estes dados reforçam as predições de modelos tridimensionais da proteína LepA388 e de outros membros da família DUF_61, as quais identificam domínios semelhantes a toxinas (como abrina e CARDS) responsáveis pela ligação e internalização celulares nos hospedeiros. Dados recentes sugerem um possível papel citotóxico desempenhado pelas proteínas desta família em leptospiras, as quais podem também ser consideradas potenciais candidatas vacinais e para diagnóstico da leptospirose, devido à sua distribuição restrita em espécies e cepas patogênicas de importância para saúde humana.

Leptospira spp. constitutes a group of gram-negative spirochete bacteria comprising saprophytic, intermediate and pathogenic species, the last being causative agents of leptospirosis, a zoonotic disease of worldwide extent and endemic in developing tropical regions. The growing number of identified leptospiral species further highlights their genetic diversity and unique virulence mechanisms, many of them with unknown function. Efforts to develop new vaccines with cross-protection and long-lasting effect have revealed possible vaccine candidates that need to be properly validated. Therefore, there is still an urgent need for a universal vaccine against leptospirosis capable of controlling and reducing the increasing outbreaks of the disease. Adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines against leptospirosis, as well as for the development of faster and more accurate diagnostic methods. Previously, three conserved hypothetical proteins in L. interrogans were identified by phage display technique, arbitrarily named as LepA069, LepA962 and LepA388. Expression of the LepA069 encoding gene showed an increase of approximately 70 % in animals infected by virulent leptospires, representing the first functional evidence of this still unknown protein. Recombinant portions of the hypothetical lipoprotein LepA962 (LepA962_Nt and LepA962_Phg) were obtained, demonstrating the strong interaction of the LepA962_Phg protein, containing the sequence identified by phage display, with laminin, plasma fibronectin, collagen I and fibrinogen in a dose-dependent manner. Furthermore, LepA962_Phg showed binding to VERO cells and its secreted extracellular matrix, and the serum obtained from this recombinant protein was able to bind to the surface of virulent leptospires, indicating that LepA962_Phg may represent an important domain of interaction between leptospires and its host. Finally, LepA388 protein belonging to an extensive family of virulence modifying proteins with unknown function (DUF_61), present only in the most virulent pathogenic leptospires, showed an increase in the expression of its encoding gene in animals infected by virulent leptospires according to data in literature. Moreover, recombinant portions of the N-terminal region of this protein showed binding to laminin, collagens I and IV, vitronectin and plasma and cell fibronectins, especially considering the sequence identified by phage display. These data support the predictions of three-dimensional models of the LepA388 protein and other members of the DUF_61 family, which identify toxin-like domains (such as abrin and CARDS) responsible for cellular binding and internalization in hosts. Recent data suggest a possible cytotoxic role played by proteins of this family in leptospires, which can also be considered potential vaccine candidates and antigens for diagnosis, due to their restricted distribution in pathogenic species and strains of importance to human health

Phase Variation in HMW1A Controls a Phenotypic Switch in Haemophilus influenzae Associated with Pathoadaptation during Persistent Infection.

Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth.

Utilizing Whole Fusobacterium Genomes To Identify, Correct, and Characterize Potential Virulence Protein Families.

Fusobacterium spp. are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including a link between the oral pathogen Fusobacterium nucleatum and the progression and severity of colorectal cancer. The identification and characterization of virulence factors in the genus Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium, we report the identification and corrected annotation of verified and potential virulence factors from the type 5 secreted autotransporter, FadA, and MORN2 protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and type strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we used sequence similarity networks to identify protein subsets and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have identified unique subsets of type 5a autotransporters, which are key proteins associated with virulence in F. nucleatum However, we coupled our bioinformatic data with bacterial binding assays to show that a predicted weakly invasive strain of F. necrophorum that lacks a Fap2 autotransporter adhesin strongly binds human colonocytes. These analyses confirm a gap in our understanding of how autotransporters, MORN2 domain proteins, and FadA adhesins contribute to host interactions and invasion. In summary, we identify candidate virulence genes in Fusobacterium, and caution that experimental validation of host-microbe interactions should complement bioinformatic predictions to increase our understanding of virulence protein contributions in Fusobacterium infections and disease.IMPORTANCEFusobacterium spp. are emerging pathogens that contribute to mammalian and human diseases, including colorectal cancer. Despite a validated connection with disease, few proteins have been characterized that define a direct molecular mechanism for Fusobacterium pathogenesis. We report a comprehensive examination of virulence-associated protein families in multiple Fusobacterium species and show that complete genomes facilitate the correction and identification of multiple, large type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell interaction experiments to show that previously predicted noninvasive strains can indeed bind to and potentially invade human cells and that this could be due to the expansion of specific virulence proteins that drive Fusobacterium infections and disease.

Streptococcus mutans serotypes and collagen-binding proteins Cnm/Cbm in children with caries analysed by PCR.

Streptococcus mutans, a primary bacterium associated with dental caries, has four known clinical serotypes (c, e, fand k). Certain serotypes, the presence of multiple serotypes and strains with collagen-binding proteins (CBP, Cnm and Cbm) have been linked with systemic disease. Evaluation of S mutans serotype distribution and caries association is needed in the United States. The purpose of this study was to evaluate the prevalence of S mutans serotypes from two cohorts of African-American children in rural Alabama using three sample types (saliva, plaque and individual S mutans isolates) by PCR detection for association with caries. Detection of CBP was also performed by PCR. In total, 129 children were evaluated and overall prevalence of serotypes were: serotype c(98%), e(26%), f(7%) and k(52%). Serotype c was statistically associated with higher caries scores in older children (P < 0.001) and serotype k was statistically more likely in females (P = 0.004). Fourteen per cent of children had CBP. Thirteen S mutans isolates from five children tested positive for both CBP. This study is the first to report on the prevalence of S mutans serotypes in a US population using the PCR-based approach. The frequency of serotype k in this study is the highest reported in any population, illustrating the need for further study to determine the prevalence of this clinically relevant serotype in the US. This is the first study to report S mutans isolates with both Cnm and Cbm in the same strain, and further analysis is needed to determine the clinical significance of these strains.

Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis.

The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Šresolution) and AtfE (1.58 Šresolution) from two P. mirabilis fimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding of P. mirabilis fimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.

A structural overview of mycobacterial adhesins: Key biomarkers for diagnostics and therapeutics.

Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, which are attractive pharmacotherapeutic targets. A large arsenal of pilus and non-pilus adhesins have been identified in mycobacteria. These adhesins are capable of interacting with host cells, including macrophages and epithelial cells and are essential to microbial pathogenesis. In the last decade, several structures of mycobacterial adhesins responsible for adhesion to either macrophages or extra cellular matrix proteins have been elucidated. In addition, key structural and functional information have emerged for the process of mycobacterial adhesion to epithelial cells, mediated by the Heparin-binding hemagglutinin (HBHA). In this review, we provide an overview of the structural and functional features of mycobacterial adhesins and discuss their role as important biomarkers for diagnostics and therapeutics. Based on the reported data, it appears clear that adhesins are endowed with a variety of different structures and functions. Most adhesins play important roles in the cell life of mycobacteria and are key virulence factors. However, they have adapted to an extracellular life to exert a role in host-pathogen interaction. The type of interactions they form with the host and the adhesin regions involved in binding is partly known and is described in this review.

Streptococcus Mutans Adhesin Biotypes that Match and Predict Individual Caries Development.

Dental caries, which affects billions of people, is a chronic infectious disease that involves Streptococcus mutans, which is nevertheless a poor predictor of individual caries development. We therefore investigated if adhesin types of S.mutans with sucrose-independent adhesion to host DMBT1 (i.e. SpaP A, B or C) and collagen (i.e. Cnm, Cbm) match and predict individual differences in caries development. The adhesin types were measured in whole saliva by qPCR in 452 12-year-old Swedish children and related to caries at baseline and prospectively at a 5-year follow-up. Strains isolated from the children were explored for genetic and phenotypic properties. The presence of SpaP B and Cnm subtypes coincided with increased 5-year caries increment, and their binding to DMBT1 and saliva correlated with individual caries scores. The SpaP B subtypes are enriched in amino acid substitutions that coincided with caries and binding and specify biotypes of S. mutans with increased acid tolerance. The findings reveal adhesin subtypes of S. mutans that match and predict individual differences in caries development and provide a rationale for individualized oral care.

Antigen I/II encoded by integrative and conjugative elements of Streptococcus agalactiae and role in biofilm formation.

Streptococcus agalactiae (i.e. Group B streptococcus, GBS) is a major human and animal pathogen. Genes encoding putative surface proteins and in particular an antigen I/II have been identified on Integrative and Conjugative Elements (ICEs) found in GBS. Antigens I/II are multimodal adhesins promoting colonization of the oral cavity by streptococci such as Streptococcus gordonii and Streptococcus mutans. The prevalence and diversity of antigens I/II in GBS were studied by a bioinformatic analysis. It revealed that antigens I/II, which are acquired by horizontal transfer via ICEs, exhibit diversity and are widespread in GBS, in particular in the serotype Ia/ST23 invasive strains. This study aimed at characterizing the impact on GBS biology of proteins encoded by a previously characterized ICE of S. agalactiae (ICE_515_tRNA(Lys)). The production and surface exposition of the antigen I/II encoded by this ICE was examined using RT-PCR and immunoblotting experiments. Surface proteins of ICE_515_tRNA(Lys) were found to contribute to GBS biofilm formation and to fibrinogen binding. Contribution of antigen I/II encoded by SAL_2056 to biofilm formation was also demonstrated. These results highlight the potential for ICEs to spread microbial adhesins between species.

Virulence characterization of non-O157 Shiga toxin-producing Escherichia coli isolates from food, humans and animals.

A total of 359 non-O157 STEC isolates from food, humans and animals were examined for serotypes, Shiga toxin subtypes and intimin subtypes. Isolates solely harboring stx2 from the three sources were selected for Vero cell cytotoxicity test. stx subtypes in eae negative isolates were more diverse than in eae positive isolates primarily carrying stx2a. Four eae subtypes (eaeß,eaeε1,eaeγ1 and eaeγ2/θ) were observed and correlated with serotypes and flagella. Food isolates showed more diverse serotypes, virulence factors and cell cytotoxicities than human isolates. Some isolates from produce belonged to serotypes that have been implicated in human diseases, carried stx2a or/and stx2dact and exhibited high cell cytotoxicity similar to human isolates. This indicates that foods can be contaminated with potentially pathogenic STEC isolates that may cause human diseases. Given the increased produce consumption and growing burden of foodborne outbreaks due to produce, produce safety should be given great importance.

Neisseria adhesin A variation and revised nomenclature scheme.

Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria meningitidis into host tissues, is one of the major components of Bexsero, a novel multicomponent vaccine licensed for protection against meningococcal serogroup B in Europe, Australia, and Canada. NadA has been identified in approximately 30% of clinical isolates and in a much lower proportion of carrier isolates. Three protein variants were originally identified in invasive meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates either lacked the gene or harbored a different variant, NadA-4. Further analysis of isolates belonging to the sequence type 213 (ST-213) clonal complex identified NadA-5, which was structurally similar to NadA-4, but more distantly related to NadA-1, -2, and -3. At the time of this writing, more than 89 distinct nadA allele sequences and 43 distinct peptides have been described. Here, we present a revised nomenclature system, taking into account the complete data set, which is compatible with previous classification schemes and is expandable. The main features of this new scheme include (i) the grouping of the previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii) the grouping of the previously assigned NadA-4 and NadA-5 variants into a single NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and (iv) the classification of the variants into two main groups, named groups I and II. To facilitate querying of the sequences and submission of new allele sequences, the nucleotide and amino acid sequences are available at http://pubmlst.org/neisseria/NadA/.

Identification of novel adhesins of M. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis.

Pathogenic bacteria interacting with eukaryotic host express adhesins on their surface. These adhesins aid in bacterial attachment to the host cell receptors during colonization. A few adhesins such as Heparin binding hemagglutinin adhesin (HBHA), Apa, Malate Synthase of M. tuberculosis have been identified using specific experimental interaction models based on the biological knowledge of the pathogen. In the present work, we carried out computational screening for adhesins of M. tuberculosis. We used an integrated computational approach using SPAAN for predicting adhesins, PSORTb, SubLoc and LocTree for extracellular localization, and BLAST for verifying non-similarity to human proteins. These steps are among the first of reverse vaccinology. Multiple claims and attacks from different algorithms were processed through argumentative approach. Additional filtration criteria included selection for proteins with low molecular weights and absence of literature reports. We examined binding potential of the selected proteins using an image based ELISA. The protein Rv2599 (membrane protein) binds to human fibronectin, laminin and collagen. Rv3717 (N-acetylmuramoyl-L-alanine amidase) and Rv0309 (L,D-transpeptidase) bind to fibronectin and laminin. We report Rv2599 (membrane protein), Rv0309 and Rv3717 as novel adhesins of M. tuberculosis H37Rv. Our results expand the number of known adhesins of M. tuberculosis and suggest their regulated expression in different stages.

New MLSB resistance gene erm(43) in Staphylococcus lentus.

The search for a specific rRNA methylase motif led to the identification of the new macrolide, lincosamide, and streptogramin B resistance gene erm(43) in Staphylococcus lentus. An inducible resistance phenotype was demonstrated by cloning and expressing erm(43) and its regulatory region in Staphylococcus aureus. The erm(43) gene was detected in two different DNA fragments, of 6,230 bp and 1,559 bp, that were each integrated at the same location in the chromosome in several S. lentus isolates of human, dog, and chicken origin.

Intestinal mucosa adherence and cytotoxicity of a sorbitol-fermenting, Shiga-toxin-negative Escherichia coli O157:NM isolate with an atypical type III secretion system.

Reports show that sorbitol-fermenting (SF) Escherichia coli O157 isolates are implicated in animal and human diseases and may represent new emerging pathogens. We investigated the cytotoxicity and interaction with intestinal tissues of an SF, Shiga-toxin-negative E. coli O157:NM isolate. This bovine isolate was negative for stx genes and was not cytotoxic for Vero cells. We found that this E. coli O157 isolate possesses an intimin of type beta, whereas the translocated intimin receptor Tir and type III secretion system components EspA, EspB, and EspD were of type alpha. In contrast, Shiga-toxin-positive O157:H7 isolates usually possess variants of type gamma. The isolate did not present typical O157:H7 attaching and effacing lesions in the newborn pig ileal in vitro organ culture model. However, extensive effacement and elongation of the microvilli were observed. In vitro organ culture results suggest that such an SF, Shiga-toxin-negative O157:NM isolate found in cattle may potentially cause disease, such as diarrhea without hemolytic uremic syndrome, in humans.

Genetic profiles of Shiga toxin and intimin genes found in stool broth cultures: a 2-year reference laboratory study.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are associated with potentially serious illness in humans. STEC detection is often based on the presence of Stxs, Stx(1) and/or Stx(2), and intimin, encoded by the eae gene. A 2-year collection of stool broth cultures was tested for variants of stx(1), stx(2), and eae. Approximately 80% (138 of 174) were positive for stx(1) and/or stx(2), with stx(1) as the most prevalent (66%). Of the stx(1) variants, stx(1) was the most common (76%) followed by stx(1c) (22%). Analysis of stx(2)-positive isolates found 20 (53%) stx(2), 13 (34%) stx(2)/stx(2v-ha), 3 (8%) stx(2v-ha), 1 (3%) stx(2v-hb), and 1 (3%) stx(2d-activatable). Findings of stx(2)/stx(2v-ha) and stx(2d-activatable) are noteworthy given associations with hemolytic uremic syndrome and increased cytotoxicity, respectively. Of the Stx positive, 94 (68%) were eae positive with 31 (33%) eae(varepsilon1), 19 (20%) eae(gamma1), and 18 (19%) eae(beta1). A predominance of eae(varepsilon1) may suggest a new pathogenic significance because, reportedly, eae(beta1) is one of the most widespread variants.

Variants of eae and stx genes of atypical enteropathogenic Escherichia coli and non-O157 Shiga toxin-producing Escherichia coli from calves.

AIMS: To determine the subtypes of stx and eae genes of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and to ascertain the typical and atypical nature of EPEC. METHODS AND RESULTS: One hundred and eighty-seven faecal samples from 134 diarrhoeic and 53 healthy calves were investigated for the presence of stx, eae and ehxA virulence genes by polymerase chain reaction and enzyme-linked immunosorbent assay. Subtype analysis of stx(1) exhibited stx(1c) in 13 (31.70%) isolates, while that of stx(2) revealed stx(2c) in eight (24.24%) and stx(2d) in two (6.06%) isolates. Subtyping of eae gene showed the presence of eae-beta, eae-eta and eae-zeta in two, three and four isolates respectively. None of the E. coli isolates possessed stx(2e), stx(2f), eae-alpha, eae-delta, eae-epsilon and eae-xi. All EPEC isolates were atypical. CONCLUSIONS: stx(1), stx(1c), stx(2), stx(2c), stx(2d), eae-beta, eae-eta and eae-zeta subtypes are prevalent in STEC and EPEC isolates in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first subtype analysis of stx(2) and eae genes of animal E. coli isolates in India and emphasizes the need to investigate their transmission to humans.

Binding of Streptococcus gordonii to extracellular matrix proteins.

Knock-out mutants of Streptococcus gordonii Challis were constructed and assayed for binding to extracellular matrix proteins (EMPs) by enzyme-linked immunosorbent assay (ELISA). It was shown that (i) the mutant lacking the cell wall polysaccharide receptor could no longer bind type I and type II collagen, (ii) the mutant lacking the fibronectin-binding proteins CshA and FbpA was also strongly impaired in collagen binding and (iii) the mutant lacking the methionine sulfoxide reductase MsrA was significantly impaired in fibronectin binding. Our results indicate that binding to EMPs by S. gordonii is a multifactorial process controlled by genes located at three different chromosomal sites.

Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin variants (muB and xiR/beta2B).

A total of 71 enteropathogenic Escherichia coli (EPEC) strains isolated from children with diarrhoea in Montevideo, Uruguay, were characterized in this study. PCR showed that 57 isolates carried eae and bfp genes (typical EPEC strains), and 14 possessed only the eae gene (atypical EPEC strains). These EPEC strains belonged to 21 O : H serotypes, including eight novel serotypes not previously reported among human EPEC in other studies. However, 72% belonged to only four serotypes: O55:H- (six strains), O111:H2 (13 strains), O111:H- (14 strains) and O119:H6 (18 strains). Nine intimin types, namely, alpha1 (two O142 strains), beta1 (29 strains, including 13 O111:H2 and 14 O111:H-), gamma1 (three O55:H- strains), theta (five strains, including three strains with H40 antigen), kappa (two strains), epsilon1 (one strain), lambda (one strain), muB (six strains of serotypes O55:H51 and O55:H-) and xiR/beta2B (22 strains, including 18 O119:H6) were detected among the 71 EPEC strains. The authors have identified two novel intimin genes (muB and xiR/beta2B) in typical EPEC strains of serotypes O55:H51/H- and O119:H6/H-. The complete nucleotide sequences of the novel muB and xiR/beta2 variant genes were determined. PFGE typing after XbaI DNA digestion was performed on 44 representative EPEC strains. Genomic DNA fingerprinting revealed 44 distinct restriction patterns and the strains were clustered in 12 groups. Only 15 strains clustered in six groups of closely related (similarity>85%) PFGE patterns, suggesting the prevailing clonal diversity among EPEC strains isolated from children with diarrhoea in Montevideo.

Opacity-associated adhesin repertoire in hyperinvasive Neisseria meningitidis.

The opacity (Opa) proteins mediate a variety of interactions between the bacterium Neisseria meningitidis and its human host. These interactions are thought to be of central importance in both the asymptomatic colonization of the nasopharynx and the sporadic occurrence of meningococcal disease. The receptor specificities of a limited number of Opa protein variants have been explored, but the high level of amino acid sequence diversity among variants has complicated the assignment of specific roles to individual Opa variants or combinations of variants. In addition, the distribution of Opa protein variants among diverse meningococci, information that is potentially informative for studies of Opa function, is poorly understood. A systematic survey of the genetic diversity in the four opa gene loci in each of 77 meningococcal isolates was undertaken. These isolates were representative of the seven hyperinvasive meningococcal clonal complexes that caused the majority of meningococcal disease over the last 50 years. Consistent with previous studies, a high level of sequence diversity was observed among the opa genes and the proteins that they encoded; however, particular sets of Opa protein variants were consistently associated with each of the clonal complexes over time periods often spanning decades and during global spread. These observations were consistent with the postulate that particular combinations of Opa proteins confer fitness advantages to individual clonal complexes and have implications for studies of Opa function and the inclusion of Opa proteins in novel meningococcal vaccines.

Culture-independent molecular subtyping of Mycoplasma pneumoniae in clinical samples.

A new molecular subtyping approach was developed which is based on the amplification and sequencing of a repetitive region of the P1 gene of Mycoplasma pneumoniae. It allows the differentiation of all known subtypes and variants of M. pneumoniae as well as the identification of new subtypes directly in clinical samples to characterize endemic and epidemic M. pneumoniae infections.