The C-Terminal Domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC Is a Lectin-Like Carbohydrate Binding Module

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbCCT) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbCCT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbCCT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. Author Summary Top Tuberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, burdens large swaths of the world population. Treatment of active TB typically requires administration of an antibiotic cocktail over several months that includes the drug ethambutol. This front line compound inhibits a set of arabinosyltransferase enzymes, called EmbA, EmbB and EmbC, which are critical for the synthesis of arabinan, a vital polysaccharide in the pathogen's unique cell envelope. How precisely ethambutol inhibits arabinosyltransferase activity is not clear, in part because structural information of its pharmacological targets has been elusive. Here, we report the high-resolution structure of the C-terminal domain of the ethambutol-target EmbC, a 390-amino acid fragment responsible for acceptor substrate recognition. Combining the X-ray crystallographic analysis with structural comparisons, site-directed mutagenesis, activity and ligand binding assays, we identified two regions in the C-terminal domain of EmbC that are capable of binding acceptor substrate mimics and are critical for activity of the full-length enzyme. Our results begin to define structure-function relationships in a family of structurally uncharacterised membrane-embedded glycosyltransferases, which are an important target for tuberculosis therapy

High Resolution Discrimination of Clinical Mycobacterium tuberculosis Complex Strains Based on Single Nucleotide Polymorphisms

Recently, the diversity of the Mycobacterium tuberculosis complex (MTBC) population structure has been described in detail. Based on geographical separation and specific host pathogen co-evolution shaping MTBC virulence traits, at least 20 major lineages/genotypes have evolved finally leading to a clear influence of strain genetic background on transmissibility, clinical presentation/outcome, and resistance development. Therefore, high resolution genotyping for characterization of strains in larger studies is mandatory for understanding mechanisms of host-pathogen-interaction and to improve tuberculosis (TB) control. Single nucleotide polymorphisms (SNPs) represent the most reliable markers for lineage classification of clinical isolates due to the low levels of homoplasy, however their use is hampered either by low discriminatory power or by the need to analyze a large number of genes to achieve higher resolution. Therefore, we carried out de novo sequencing of 26 genes (approx. 20000 bp per strain) in a reference collection of MTBC strains including all major genotypes to define a highly discriminatory gene set. Overall, 161 polymorphisms were detected of which 59 are genotype-specific, while 13 define deeper branches such as the Euro-American lineage. Unbiased investigation of the most variable set of 11 genes in a population based strain collection (one year, city of Hamburg, Germany) confirmed the validity of SNP analysis as all strains were classified with high accuracy. Taken together, we defined a diagnostic algorithm which allows the identification of 17 MTBC phylogenetic lineages with high confidence for the first time by sequencing analysis of just five genes. In conclusion, the diagnostic algorithm developed in our study is likely to open the door for a low cost high resolution sequence/SNP based differentiation of the MTBC with a very high specificity. High throughput assays can be established which will be needed for large association studies that are mandatory for detailed investigation of host-pathogen-interaction during TB infection

Inhibition of IL-10 Production by Maternal Antibodies against Group B Streptococcus GAPDH Confers Immunity to Offspring by Favoring Neutrophil Recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10−/−) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion

Drug resistance mutations and heteroresistance detected using the GenoType MTBDRplus assay and their implication for treatment outcomes in patients from Mumbai, India

<p>Abstract</p> <p>Background</p> <p>Only 5% of the estimated global multidrug resistant TB (MDRTB) load is currently detected. Endemic Mumbai with increasing MDR would benefit from the introduction of molecular methods to detect resistance.</p> <p>Methods</p> <p>The GenoType MTBDR<it>plus </it>assay was used to determine mutations associated with isoniazid and rifampicin resistance and their correlation with treatment outcomes. It was performed on a convenience sample comprising 88 onset and 67 fifth month isolates for which phenotypic drug susceptibility testing (DST) was determined by the Buddemeyer technique for an earlier study. Simultaneous presence of wild type and mutant bands was referred to as "mixed patterns" (heteroresistance).</p> <p>Results</p> <p>Phenotypically 41 isolates were sensitive; 11 isoniazid, 2 rifampicin, 2 pyrazinamide and 5 ethambutol monoresistant; 16 polyresistant and 78 MDR. The agreement between both methods was excellent (kappa = 0.72-0.92). Of 22 rifampicin resistant onset isolates, the predominant <it>rpoB </it>mutations were the singular lack of WT8 (n = 8) and mixed D516V patterns (n = 9). Of the 64 rifampicin resistant fifth month isolates, the most frequent mutations were in WT8 (n = 31) with a further 9 showing the S531L mutation. Mixed patterns were seen in 22 (34%) isolates, most frequently for the D516V mutation (n = 21). Of the 22 onset and 35 fifth month <it>katG </it>mutants, 13 and 12 respectively showed the S315T1 mutation with loss of the WT. Mixed patterns involving both S315T1 and S315T2 were seen in 9 and 23 isolates respectively. Seventeen of 23 and 23/35 <it>inhA </it>mutant onset and fifth month isolates showed mixed A16G profiles. Additionally, 10 fifth month isolates lacked WT2. Five onset and 6 fifth month isolates had both <it>katG </it>and <it>inhA </it>mutations. An association was noted between only <it>katG </it>but not only <it>inhA </it>resistance and poor outcome (<it>p </it>= 0.037); and additional resistance to ethambutol (<it>p </it>= 0.0033). More fifth month than onset isolates had mixed profiles for at least 1 gene (<it>p </it>= 0.000001).</p> <p>Conclusions</p> <p>The use of the assay to rapidly diagnose MDR could guide simultaneous first- and second-line DST, and reduce the delay in administering appropriate regimens. Furthermore, detection of heteroresistance could prevent inaccurate "cured" treatment outcomes documented through smear microscopy and permit more sensitive detection of neonascent resistance.</p

High-Resolution Melting Analysis for the Rapid Detection of Fluoroquinolone and Streptomycin Resistance in Mycobacterium tuberculosis

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Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution

<p>Abstract</p> <p>Background</p> <p>Group B <it>Streptococcus </it>(GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations.</p> <p>Methods</p> <p>To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence.</p> <p>Results</p> <p>Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005).</p> <p>Conclusion</p> <p>This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.</p

Isoniazid resistance levels of Mycobacterium tuberculosis can largely be predicted by high-confidence resistance-conferring mutations.

YesThe majority of Mycobacterium tuberculosis isolates resistant to isoniazid harbour a mutation in katG. Since these mutations cause a wide range of minimum inhibitory concentrations (MICs), largely below the serum level reached with higher dosing (15 mg/L upon 15–20 mg/kg), the drug might still remain partly active in presence of a katG mutation. We therefore investigated which genetic mutations predict the level of phenotypic isoniazid resistance in clinical M. tuberculosis isolates. To this end, the association between known and unknown isoniazid resistance-conferring mutations in whole genome sequences, and the isoniazid MICs of 176 isolates was examined. We found mostly moderate-level resistance characterized by a mode of 6.4 mg/L for the very common katG Ser315Thr mutation, and always very high MICs (≥19.2 mg/L) for the combination of katG Ser315Thr and inhA c-15t. Contrary to common belief, isolates harbouring inhA c-15t alone, partly also showed moderate-level resistance, particularly when combined with inhA Ser94Ala. No overt association between low-confidence or unknown mutations, except in katG, and isoniazid resistance (level) was found. Except for the rare katG deletion, line probe assay is thus not sufficiently accurate to predict the level of isoniazid resistance for a single mutation in katG or inhA.European Research Council (Starting Grant INTERRUPTB 311725 to CM, LR and BdJ), The Damien Foundatio