Mycobacterium tuberculosis Exploits Asparagine to Assimilate Nitrogen and Resist Acid Stress during Infection

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.     

Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.     

Mycobacterium tuberculosis Beijing Genotype Is Associated with HIV Infection in Mozambique

The Beijing genotype is a lineage of Mycobacterium tuberculosis that is distributed worldwide and responsible for large epidemics, associated with multidrug-resistance. However, its distribution in Africa is less understood due to the lack of data. Our aim was to investigate the prevalence and possible transmission of Beijing strains in Mozambique by a multivariate analysis of genotypic, geographic and demographic data. A total of 543 M. tuberculosis isolates from Mozambique were spoligotyped. Of these, 33 were of the Beijing lineage. The genetic relationship between the Beijing isolates were studied by identification of genomic deletions within some Regions of Difference (RD), Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetivie Unit – variable number tandem repeat (MIRU-VNTR). Beijing strains from South Africa, representing different sublineages were included as reference strains. The association between Beijing genotype, Human Immunodeficiency Virus (HIV) serology and baseline demographic data was investigated. HIV positive serostatus was significantly (p=0.023) more common in patients with Beijing strains than in patients with non-Beijing strains in a multivariable analysis adjusted for age, sex and province (14 (10.9%) of the 129 HIV positive patients had Beijing strains while 6/141 (4.3%) of HIV negative patients had Beijing strains). The majority of Beijing strains were found in the Southern region of Mozambique, particularly in Maputo City (17%). Only one Beijing strain was drug resistant (multi-drug resistant). By combined use of RD and spoligotyping, three genetic sublineages could be tentatively identified where a distinct group of four isolates had deletion of RD150, a signature of the “sublineage 7” recently emerging in South Africa. The same group was very similar to South African “sublineage 7” by RFLP and MIRU-VNTR, suggesting that this sublineage could have been recently introduced in Mozambique from South Africa, in association with HIV infection.     

Mycobacterium tuberculosis Is a Natural Ornithine Aminotransferase (rocD) Mutant and Depends on Rv2323c for Growth on Arginine

Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using ¹³C- or ¹⁵N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb.     

Mycobacterium tuberculosis Infection in Young Children: Analyzing the Performance of the Diagnostic Tests

Objective

This study evaluated the performance of the Tuberculin Skin Test (TST) and Quantiferon-TB Gold in-Tube (QFT) and the possible association of factors which may modify their results in young children (0–6 years) with recent contact with an index tuberculosis case.

Materials and Methods

A cross-sectional study including 135 children was conducted in Manaus, Amazonas-Brazil. The TST and QFT were performed and the tests results were analyzed in relation to the personal characteristics of the children studied and their relationship with the index case.

Results

The rates of positivity were 34.8% (TST) and 26.7% (QFT), with 14.1% of indeterminations by the QFT. Concordance between tests was fair (Kappa = 0.35 P<0.001). Both the TST and QFT were associated with the intensity of exposure (Linear OR = 1.286, P = 0.005; Linear OR = 1.161, P = 0.035 respectively) with only the TST being associated with the time of exposure (Linear OR = 1.149, P = 0.009). The presence of intestinal helminths in the TST+ group was associated with negative QFT results (OR = 0.064, P = 0.049). In the TST− group lower levels of ferritin were associated with QFT+ results (Linear OR = 0.956, P = 0.036).

Conclusions

Concordance between the TST and QFT was lower than expected. The factors associated with the discordant results were intestinal helminths, ferritin levels and exposure time to the index tuberculosis case. In TST+ group, helminths were associated with negative QFT results suggesting impaired cell-mediated immunity. The TST−&QFT+ group had a shorter exposure time and lower ferritin levels, suggesting that QFT is faster and ferritin may be a potential biomarker of early stages of tuberculosis infection.     

Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications.     

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser³¹⁵ which are present in approximately 50–90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser³¹⁵ appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser³¹⁵ mutations are favoured in the slow growing cultures.     

Trans-Translation in Helicobacter pylori: Essentiality of Ribosome Rescue and Requirement of Protein Tagging for Stress Resistance and Competence

Background

The ubiquitous bacterial trans-translation is one of the most studied quality control mechanisms. Trans-translation requires two specific factors, a small RNA SsrA (tmRNA) and a protein co-factor SmpB, to promote the release of ribosomes stalled on defective mRNAs and to add a specific tag sequence to aberrant polypeptides to direct them to degradation pathways. Helicobacter pylori is a pathogen persistently colonizing a hostile niche, the stomach of humans.

Principal Findings

We investigated the role of trans-translation in this bacterium well fitted to resist stressful conditions and found that both smpB and ssrA were essential genes. Five mutant versions of ssrA were generated in H. pylori in order to investigate the function of trans-translation in this organism. Mutation of the resume codon that allows the switch of template of the ribosome required for its release was essential in vivo, however a mutant in which this codon was followed by stop codons interrupting the tag sequence was viable. Therefore one round of translation is sufficient to promote the rescue of stalled ribosomes. A mutant expressing a truncated SsrA tag was viable in H. pylori, but affected in competence and tolerance to both oxidative and antibiotic stresses. This demonstrates that control of protein degradation through trans-translation is by itself central in the management of stress conditions and of competence and supports a regulatory role of trans-translation-dependent protein tagging. In addition, the expression of smpB and ssrA was found to be induced upon acid exposure of H. pylori.

Conclusions

We conclude to a central role of trans-translation in H. pylori both for ribosome rescue possibly due to more severe stalling and for protein degradation to recover from stress conditions frequently encountered in the gastric environment. Finally, the essential trans-translation machinery of H. pylori is an excellent specific target for the development of novel antibiotics.     

glpx Gene in Mycobacterium tuberculosis Is Required for In Vitro Gluconeogenic Growth and In Vivo Survival

Several enzymes involved in central carbon metabolism and gluconeogenesisplay a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb). The only known functional fructose 1,6-bisphosphatase (FBPase) in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2–3 log₁₀ lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.     

O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.     

The FgHOG1 Pathway Regulates Hyphal Growth,Stress Responses,and Plant Infection in Fusarium graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley worldwide. In a previous study of systematic characterization of protein kinase genes in F. graminearum, mutants of three putative components of the osmoregulation MAP kinase pathway were found to have distinct colony morphology and hyphal growth defects on PDA plates. Because the osmoregulation pathway is not known to regulate aerial hyphal growth and branching, in this study we further characterized the functions of the FgHog1 pathway in growth, pathogenesis, and development. The Fghog1, Fgpbs2, and Fgssk2 mutants were all reduced in growth rate, aerial hyphal growth, and hyphal branching angle. These mutants were not only hypersensitive to osmotic stress but also had increased sensitivity to oxidative, cytoplasm membrane, and cell wall stresses. The activation of FgHog1 was blocked in the Fgpbs2 and Fgssk2 mutants, indicating the sequential activation of FgSsk2-FgPbs2-FgHog1 cascade. Interestingly, the FgHog1 MAPK pathway mutants appeared to be sensitive to certain compounds present in PDA. They were female sterile but retained male fertility. We also used the metabolomics profiling approach to identify compatible solutes that were accumulated in the wild type but not in the Fghog1 deletion mutant. Overall, our results indicate that the FgSsk2-FgPbs2-FgHog1 MAPK cascade is important for regulating hyphal growth, branching, plant infection, and hyperosmotic and general stress responses in F. graminearum.     

Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage.     

Caenorhabditis elegans: A Simple Nematode Infection Model for Penicillium marneffei

Penicillium marneffei, one of the most important thermal dimorphic fungi, is a severe threat to the life of immunocompromised patients. However, the pathogenic mechanisms of P. marneffei remain largely unknown. In this work, we developed a model host by using nematode Caenorhabditis elegans to investigate the virulence of P. marneffei. Using two P. marneffei clinical isolate strains 570 and 486, we revealed that in both liquid and solid media, the ingestion of live P. marneffei was lethal to C. elegans (P<0.001). Meanwhile, our results showed that the strain 570, which can produce red pigment, had stronger pathogenicity in C. elegans than the strain 486, which can’t produce red pigment (P<0.001). Microscopy showed the formation of red pigment and hyphae within C. elegans after incubation with P. marneffei for 4 h, which are supposed to be two contributors in nematodes killing. In addition, we used C. elegans as an in vivo model to evaluate different antifungal agents against P. marneffei, and found that antifungal agents including amphotericin B, terbinafine, fluconazole, itraconazole and voriconazole successfully prolonged the survival of nematodesinfected by P. marneffei. Overall, this alternative model host can provide us an easy tool to study the virulence of P. marneffei and screen antifungal agents.     

Yersinia pestis: New Evidence for an Old Infection

The successful reconstruction of an ancient bacterial genome from archaeological material presents an important methodological advancement for infectious disease research. The reliability of evolutionary histories inferred by the incorporation of ancient data, however, are highly contingent upon the level of genetic diversity represented in modern genomic sequences that are publicly accessible, and the paucity of available complete genomes restricts the level of phylogenetic resolution that can be obtained. Here we add to our original analysis of the Yersinia pestis strain implicated in the Black Death by consolidating our dataset for 18 modern genomes with single nucleotide polymorphism (SNP) data for an additional 289 strains at over 600 positions. The inclusion of this additional data reveals a cluster of Y. pestis strains that diverge at a time significantly in advance of the Black Death, with divergence dates roughly coincident with the Plague of Justinian (6^th to 8^th century AD). In addition, the analysis reveals further clues regarding potential radiation events that occurred immediately preceding the Black Death, and the legacy it may have left in modern Y. pestis populations. This work reiterates the need for more publicly available complete genomes, both modern and ancient, to achieve an accurate understanding of the history of this bacterium.     

Mycobacterium tuberculosis DosR is Required for Activity of the PmbtB and PmbtI Promoters under Hypoxia

Mycobacterium tuberculosis has the ability to survive for extended periods of time under conditions of low oxygen, low pH, low iron and low nutrients. The mycobactins (M. tuberculosis siderophores) play a key role in scavenging iron from the environment and are induced in response to low iron in an IdeR-regulated manner. We demonstrate that the promoters of two mycobactin gene (mbt) operons are also expressed during adaptation to low oxygen, and that this expression is dependent on the DosR regulator. Up-regulation of mbt operons induced by low iron was not DosR-dependent. DosR is a member of a two component regulatory system which responds to oxygen availability. Deletion of the DosR regulator led to increased expression of bacterioferritin and increased capacity to grow under iron depletion. These data provide a link between the mycobacterial response to two conditions likely to be encountered in vivo, low iron and low oxygen.