The Complete Genome Sequence of the Emerging Pathogen Mycobacterium haemophilum Explains Its Unique Culture Requirements

Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins or to utilize ferri-mycobactins to support growth. These differences correlate with the absence of genes associated with mycobactin synthesis, secretion, and uptake. In agreement with the ability of heme to promote growth, we identified genes encoding heme uptake machinery. Consistent with its propensity to infect the skin, we show at the whole-genome level the genetic closeness of M. haemophilumwith Mycobacterium leprae, an organism which cannot be cultivated in vitro, and we identify genes uniquely shared by these organisms. Finally, we identify means to express foreign genes in M. haemophilum. These data explain the unique culture requirements for this important pathogen, provide a foundation upon which the genome sequence can be exploited to improve diagnostics and therapeutics, and suggest use of M. haemophilum as a tool to elucidate functions of genes shared with M. leprae. IMPORTANCE Mycobacterium haemophilum is an emerging pathogen with an unknown natural reservoir that exhibits unique requirements for iron supplementation to grow in vitro. Understanding the basis for this iron requirement is important because it is fundamental to isolation of the organism from clinical samples and environmental sources. Defining the molecular basis for M. haemophilium\u27s growth requirements will also shed new light on mycobacterial strategies to acquire iron and can be exploited to define how differences in such strategies influence pathogenesis. Here, through a combination of sequencing and experimental approaches, we explain the basis for the iron requirement. We further demonstrate the genetic closeness of M. haemophilum and Mycobacterium leprae, the causative agent of leprosy which cannot be cultured in vitro, and we demonstrate methods to genetically manipulate M. haemophilum. These findings pave the way for the use of M. haemophilum as a model to elucidate functions of genes shared with M. leprae

Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. The M. luteus Rpf is a secreted ∼16-kDa protein which restores active growth to cultures of M. luteus rendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins of M. tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M. tuberculosis Rpf homologues in vivo. To address these questions, we have disrupted each of the five rpf-like genes in M. tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast to M. luteus, for which rpf is an essential gene, we find that all of the M. tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis of rpf expression in M. tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M. tuberculosis rpf genes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M. tuberculosis

A Mycobacterium tuberculosis Rpf Double-Knockout Strain Exhibits Profound Defects in Reactivation from Chronic Tuberculosis and Innate Immunity Phenotypes▿

Resuscitation-promoting factors (Rpfs), apparent peptidoglycan hydrolases, have been implicated in the reactivation of dormant bacteria. We previously demonstrated that deletion of rpfB impaired reactivation of Mycobacterium tuberculosis in a mouse model. Because M. tuberculosis encodes five Rpf paralogues, redundant functions among the family members might obscure rpf single-knockout phenotypes. A series of rpf double knockouts were therefore generated. One double mutant, ΔrpfAB, exhibited several striking phenotypes. Consistent with the proposed cell wall-modifying function of Rpfs, ΔrpfAB exhibited an altered colony morphology. Although ΔrpfAB grew comparably to the parental strain in axenic culture, in vivo it exhibited deficiency in reactivation induced in C57BL/6 mice by the administration of nitric oxide synthase inhibitor (aminoguanidine) or by CD4+ T-cell depletion. Notably, the reactivation deficiency of ΔrpfAB was more severe than that of ΔrpfB in aminoguanidine-treated mice. A similar deficiency was observed in ΔrpfAB reactivation from a drug-induced apparently sterile state in infected NOS2−/− mice upon cessation of antimycobacterial therapy. Secondly, ΔrpfAB showed a persistence defect not seen with the ΔrpfB or ΔrpfA single mutants. Interestingly, ΔrpfAB exhibited impaired growth in primary mouse macrophages and induced higher levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin 6. Simultaneous reintroduction of rpfA and rpfB into the double-knockout strain complemented the colony morphology and macrophage cytokine secretion phenotypes. Phenotypes related to cell wall composition and macrophage responses suggest that M. tuberculosis Rpfs may influence the outcome of reactivation, in part, by modulating innate immune responses to the bacterium

COVID-19 Vaccine-Induced Lichenoid Eruptions—Clinical and Histopathologic Spectrum in a Case Series of Fifteen Patients with Review of the Literature

Lichen planus is a distinctive mucocutaneous disease with well-established clinical and histopathologic criteria. Lichenoid eruptions closely resemble lichen planus and may sometimes be indistinguishable from it. Systemic agents previously associated have included medications, viral infections and vaccines. Sporadic case reports of lichen planus and lichenoid reactions associated with COVID-19 vaccines have recently emerged. Herein, we review the world literature (31 patients) and expand it with a case series of 15 patients who presented with vaccine-induced lichenoid eruption (V-ILE). The spectrum of clinical and histopathologic findings is discussed with emphasis on the subset whose lesions manifested in embryologic fusion lines termed lines of Blaschko. This rare Blaschkoid distribution appeared in seven of the 46 patients studied. Of interest, all seven were linked to the mRNA COVID-19 vaccines. We believe that all lichenoid eruptions should be approached with a heightened index of suspicion and patients should be specifically questioned with regards to their vaccination history. When diagnosed early in its course, V-ILE is easily treated and resolves quickly in almost all patients with or without hyperpigmentation. Additional investigative studies regarding its immunopathology and inflammatory signaling pathways may offer insight into other Th1-driven autoimmune phenomena related to COVID-19 vaccination

Derailing the aspartate pathway of Mycobacterium tuberculosis to eradicate persistent infection

A major constraint for developing new anti-tuberculosis drugs is the limited number of validated targets that allow eradication of persistent infections. Here, we uncover a vulnerable component of Mycobacterium tuberculosis (Mtb) persistence metabolism, the aspartate pathway. Rapid death of threonine and homoserine auxotrophs points to a distinct susceptibility of Mtb to inhibition of this pathway. Combinatorial metabolomic and transcriptomic analysis reveals that inability to produce threonine leads to deregulation of aspartate kinase, causing flux imbalance and lysine and DAP accumulation. Mtb’s adaptive response to this metabolic stress involves a relief valve-like mechanism combining lysine export and catabolism via aminoadipate. We present evidence that inhibition of the aspartate pathway at different branch-point enzymes leads to clearance of chronic infections. Together these findings demonstrate that the aspartate pathway in Mtb relies on a combination of metabolic control mechanisms, is required for persistence, and represents a target space for anti-tuberculosis drug development.ISSN:2041-172

Novel Inhibitors of InhA Efficiently Kill Mycobacterium tuberculosis under Aerobic and Anaerobic Conditions▿

Drug resistance in Mycobacterium tuberculosis has become a serious global health threat, which is now complicated by the emergence of extensively drug-resistant strains. New drugs that are active against drug-resistant tuberculosis (TB) are needed. We chose to search for new inhibitors of the enoyl-acyl carrier protein (ACP) reductase InhA, the target of the first-line TB drug isoniazid (also known as isonicotinoic acid hydrazide [INH]). A subset of a chemical library, composed of 300 compounds inhibiting Plasmodium falciparum enoyl reductase, was tested against M. tuberculosis. Four compounds were found to inhibit M. tuberculosis growth with MICs ranging from 1 μM to 10 μM. Testing of these compounds against M. tuberculosis in vitro revealed that only two compounds (CD39 and CD117) were bactericidal against drug-susceptible and drug-resistant M. tuberculosis. These two compounds were also bactericidal against M. tuberculosis incubated under anaerobic conditions. Furthermore, CD39 and CD117 exhibited increased bactericidal activity when used in combination with INH or rifampin, but CD39 was shown to be toxic to eukaryotic cells. The compounds inhibit InhA as well the fatty acid synthase type I, and CD117 was found to also inhibit tuberculostearic acid synthesis. This study provides the TB drug development community with two chemical scaffolds that are suitable for structure-activity relationship study to improve on their cytotoxicities and bactericidal activities in vitro and in vivo

Mycobacterium tuberculosis EsxH inhibits ESCRT-dependent CD4+ T-cell activation

Mycobacterium tuberculosis (Mtb) establishes a persistent infection, despite inducing antigen-specific T-cell responses. Although T cells arrive at the site of infection, they do not provide sterilizing immunity. The molecular basis of how Mtb impairs T-cell function is not clear. Mtb has been reported to block major histocompatibility complex class II (MHC-II) antigen presentation; however, no bacterial effector or host-cell target mediating this effect has been identified. We recently found that Mtb EsxH, which is secreted by the Esx-3 type VII secretion system, directly inhibits the endosomal sorting complex required for transport (ESCRT) machinery. Here, we showed that ESCRT is required for optimal antigen processing; correspondingly, overexpression and loss-of-function studies demonstrated that EsxH inhibited the ability of macrophages and dendritic cells to activate Mtb antigen-specific CD4+ T cells. Compared with the wild-type strain, the esxH-deficient strain induced fivefold more antigen-specific CD4+ T-cell proliferation in the mediastinal lymph nodes of mice. We also found that EsxH undermined the ability of effector CD4+ T cells to recognize infected macrophages and clear Mtb. These results provide a molecular explanation for how Mtb impairs the adaptive immune response

Correction: Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-Å-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection