It is often assumed that Mycobacterium tuberculosis (Mtb)-induced granulomatous lesions, particularly those undergoing central caseation, are anoxic, and that the survival of Mtb in these lesions requires the integrity of its non-oxidative respiratory pathways. Using the hypoxia marker pimonidazole, we now provide immunohistochemical evidence that in the most frequently used animal model system of inbred mice Mtb-induced granulomas, even after more than one year of aerogenic infection, are not severely hypoxic. In contrast, chronic aerosol infection with M. avium strain TMC724 was associated with hypoxia surrounding necrotizing granuloma centres. Direct measurements of oxygen tension with a flexible microelectrode in mouse lungs chronically infected with Mtb disclosed a wide range of oxygen partial pressures in different parts of the lungs which, however, rarely approached the anoxic conditions consistently found in necrotizing tumours. We further show that an Mtb mutant, defective in nitrate reductase (narG) necessary for survival under anaerobic conditions in vitro, can persist in the lungs of chronically infected mice to a similar extent as wild-type Mtb. These findings have important implications for the use of the mouse model of Mtb infection in developing eradication chemotherapy and for evaluating putative mechanisms of chronic persistence and latency of Mtb.

Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc 2 155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.

Streptococcus pneumoniae (pneumococcus) is a pathogen that causes severe local and life-threatening invasive diseases, which are associated with high mortality rates. Pneumococci have evolved several strategies to evade the host immune system, including complement to disseminate and to survive in various host niches. Thus, pneumococci bind complement inhibitors such as C4b-binding protein (C4BP) and factor H via pneumococcal surface protein C, thereby inhibiting the classical and alternative complement pathways. In this study, we identified the pneumococcal glycolytic enzyme enolase, a nonclassical cell surface and plasminogen-binding protein, as an additional pneumococcal C4BP-binding protein. Furthermore, we demonstrated that human, but not mouse, C4BP bound pneumococci. Recombinant enolase bound in a dose-dependent manner C4BP purified from plasma, and the interaction was reduced by increasing ionic strength. Enolase recruited C4BP and plasminogen, but not factor H, from human serum. Moreover, C4BP and plasminogen bound to different domains of enolase as they did not compete for the interaction with enolase. In direct binding assays with recombinant C4BP mutants lacking individual domains, two binding sites for enolase were identified on the complement control protein (CCP) domain 1/CCP2 and CCP8 of the C4BP α-chains. C4BP bound to the enolase retained its cofactor activity as determined by C4b degradation. Furthermore, in the presence of exogenously added enolase, an increased C4BP binding to and subsequently decreased C3b deposition on pneumococci was observed. Taken together, pneumococci specifically interact with human C4BP via enolase, which represents an additional mechanism of human complement control by this versatile pathogen.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.

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 125 I-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.

Tuberculosis is a leading cause of illness and death in Congo. No data are available about the population structure and transmission dynamics of the Mycobacterium tuberculosis complex strains prevalent in this central Africa country. On the basis of single-nucleotide polymorphisms detected by whole-genome sequencing, we phylogenetically characterized 74 MTBC isolates from Brazzaville, the capital of Congo. The diversity of the study population was high; most strains belonged to the Euro-American lineage, which split into Latin American Mediterranean, Uganda I, Uganda II, Haarlem, X type, and a new dominant sublineage named Congo type (n = 26). Thirty strains were grouped in 5 clusters (each within 12 single-nucleotide polymorphisms), from which 23 belonged to the Congo type. High cluster rates and low genomic diversity indicate recent emergence and transmission of the Congo type, a new Euro-American sublineage of MTBC.

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼10 2 colony forming units (CFUs) and ∼10 3 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation—roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.

LytR-cpsA-Psr (LCP) domain containing proteins fulfil important functions in bacterial cell wall synthesis. In Mycobacterium tuberculosis complex (Mtbc) strains, the causative agents of tuberculosis (TB), the genes Rv3484 and Rv3267 encode for LCP proteins which are putatively involved in arabinogalactan transfer to peptidoglycan. To evaluate the significance of Rv3484 for Mtbc virulence, we generated a deletion mutant in the Mtbc strain H37Rv and studied its survival in mice upon aerosol infection. The deletion mutant failed to establish infection demonstrating that Rv3484 is essential for growth in mice. Following an initial phase of marginal replication in the lungs until day 21, the Rv3484 deletion mutant was almost eliminated by day 180 post-infectionem. Interestingly, the mutant also showed higher levels of resistance to meropenem/clavulanate and lysozyme, both targeting peptidoglycan structure. We conclude that Rv3484 is essential for Mtbc virulence in vivo where its loss of function cannot be compensated by Rv3267.