Microevolution of extensively drug-resistant tuberculosis in Russia.

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72%belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted. © 2012 by Cold Spring Harbor Laboratory Press

Borrelia recurrentis employs a novel multifunctional surface protein with anti-complement, anti-opsonic and invasive potential to escape innate immunity

Borrelia recurrentis, the etiologic agent of louse-borne relapsing fever in humans, has evolved strategies, including antigenic variation, to evade immune defence, thereby causing severe diseases with high mortality rates. Here we identify for the first time a multifunctional surface lipoprotein of B. recurrentis, termed HcpA, and demonstrate that it binds human complement regulators, Factor H, CFHR-1, and simultaneously, the host protease plasminogen. Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack. Moreover, ectopic expression of HcpA in a B. burgdorferi B313 strain, deficient in Factor H binding proteins, protected the transformed spirochetes from complement-mediated killing. Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components. Together, the present study underscores the high virulence potential of B. recurrentis. The elucidation of the molecular basis underlying the versatile strategies of B. recurrentis to escape innate immunity and to persist in human tissues, including the brain, may help to understand the pathological processes underlying louse-borne relapsing fever

Inactivation of DltA Modulates Virulence Factor Expression in Streptococcus pyogenes

D-alanylated lipoteichoic acid is a virtually ubiquitous component of gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes.Isogenic Delta dltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of Delta dltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in Delta dltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated.This study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of gram-positive pathogens in the search for novel antimicrobial compounds

Interaction between M-Like Protein and Macrophage Thioredoxin Facilitates Antiphagocytosis for Streptococcus equi ssp. zooepidemicus

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z) is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis

Yersinia enterocolitica Serum Resistance Proteins YadA and Ail Bind the Complement Regulator C4b-Binding Protein

Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β2-glycoprotein I and interferes with innate immune recognition

Evaluation of invasive and non-invasive methods for the diagnosis of Helicobacter pylori infection in symptomatic children and adolescents

CONTEXT: Multiple diagnostic methods are available for the detection of Helicobacter pylori infection, but at present no single one can be used as the gold standard. OBJECTIVE: The aim of this study was to evaluate the diagnostic accuracy of 3 invasive and 2 non-invasive methods for detection of Helicobacter pylori infection in symptomatic children and adolescents. DESIGN: Prospective cohort study SETTING: Peptic Disease outpatients service, Discipline of Pediatric Gastroenterology, Universidade Federal de São Paulo (UNIFESP) / Escola Paulista de Medicina. PATIENTS: Forty-seven patients who underwent endoscopy because of dyspeptic symptoms. DIAGNOSTIC METHODS: Endoscopy with gastric biopsies for 3 invasive (rapid urease test, histology and culture) and 2 non-invasive methods (a commercial ELISA serology and 13carbon urea breath test - isotope ratio mass spectrometry) for detection of Helicobacter pylori infection. MAIN MEASUREMENTS: Sensitivity, specificity, positive and negative predictive values of each method and agreement and disagreement rates between the methods. RESULTS: Forty-seven patients [mean age, 11y9mo (SD 2y10mo), 27 female and 20 male]; 62% of them were Helicobacter pylori-positive. All methods agreed in 61%, and were negative in 21% and positive in 40%. The greatest concordance between 2 methods occurred between the invasive methods: histology and rapid urease test (89.6%) and histology and culture (87.5%). The greatest sensitivity, considering Helicobacter pylori-positive cases, for any combination of 3 or more tests, was achieved by the rapid urease test (S=100%), followed by histology, serology and 13carbon-urea breath test (S=93.1%) and lastly by culture (S=79.3%). The highest specificity was obtained by histology (100%) and culture (100%), followed by the rapid urease test (84.2%), serology (78.9%) and 13carbon-urea breath test (78.9%). CONCLUSIONS: Our results suggest that among invasive methods, an association between the rapid urease test and histology constituted the best choice for the detection of Helicobacter pylori infection. If results of histology and the rapid urease test are different, serology may be recommended.CONTEXTO: Vários métodos diagnósticos estão disponíveis para a detecção da infecção por Helicobacter pylori (Hp), porém, até o presente momento, não há um teste que possa ser utilizado isoladamente como padrão-ouro. OBJETIVO: Avaliar a acurácia de três métodos invasivos e dois não-invasivos na detecção da infecção por Hp em crianças e adolescentes sintomáticos. TIPO DE ESTUDO: Estudo coorte prospectivo. LOCAL: Ambulatório de Doença Péptica, Disciplina de Gastroenterologia Pediátrica, Universidade Federal de São Paulo (UNIFESP) / Escola Paulista de Medicina. PACIENTES: 47 pacientes sintomáticos que realizaram exame endoscópico devido a sintomas dispépticos. MÉTODOS DIAGNÓSTICOS: Exame endoscópico com biopsias gástricas para três métodos invasivos (teste rápido da urease, histologia e cultura) e dois métodos não-invasivos (teste sorológico ELISA industrializado e teste respiratório com uréia marcada com Carbono13). VARIÁVEIS ESTUDADAS: Sensibilidade, especificidade, valor preditivo positivo e negativo de cada método e taxas de concordância e discordância entre os métodos. RESULTADOS: 47 pacientes [idade média de 11a9m (DP 2a10m), 27 do sexo feminino e 20 do masculino], 62% deles com infecção por Hp. Todos os 5 métodos concordaram em 61%, sendo negativo em 21% e positivo em 40%. As maiores concordâncias entre dois métodos ocorreram entre os métodos invasivos: histologia e teste rápido da urease (89,6%) e entre a histologia e cultura (87,5%). A maior sensibilidade, considerando como Hp positivo, qualquer combinação de 3 ou mais testes, foi encontrada no teste rápido da urease (S=100%), seguido pela histologia, sorologia e o teste respiratório com uréia marcada com Carbono13 (S=93,1%) e por fim a cultura (S=79,3%). A maior especificidade foi obtida pela histologia e cultura (100%), seguidos pelo teste rápido da urease (84,2%), sorologia (78,9%) e teste respiratório com uréia marcada com Carbono13 (78,9%). CONCLUSÕES: Nossos resultados sugerem que, entre os métodos invasivos, a associação do teste rápido da urease e histologia constituem a melhor escolha para a detecção da infecção por Hp. Se os resultados da histologia e do teste rápido da urease forem discordantes é recomendada a sorologia.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Pediatric DepartmentUNIFESP, EPM, Pediatric DepartmentSciEL

Use of cancer-specific yeast-secreted in vivo biotinylated recombinant antibodies for serum biomarker discovery

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Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases