Mycobacterium tuberculosis, the leading cause of death due to infection, has a dynamic and immunomodulatory cell envelope. The cell envelope structurally and functionally varies across the length of ...the cell and during the infection process. This variability allows the bacterium to manipulate the human immune system, tolerate antibiotic treatment and adapt to the variable host environment. Much of what we know about the mycobacterial cell envelope has been gleaned from model actinobacterial species, or model conditions such as growth in vitro, in macrophages and in the mouse. In this Review, we combine data from different experimental systems to build a model of the dynamics of the mycobacterial cell envelope across space and time. We describe the regulatory pathways that control metabolism of the cell wall and surface lipids in M. tuberculosis during growth and stasis, and speculate about how this regulation might affect antibiotic susceptibility and interactions with the immune system.
Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced ...susceptibility to antibiotics-even below this breakpoint-is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.
MR1-restricted mucosal-associated invariant T (MAIT) cells represent a subpopulation of αβ T cells with innate-like properties and limited TCR diversity. MAIT cells are of interest because of their ...reactivity against bacterial and yeast species, suggesting that they play a role in defense against pathogenic microbes. Despite the advances in understanding MAIT cell biology, the molecular and structural basis behind their ability to detect MR1-Ag complexes is unclear. In this study, we present our structural and biochemical characterization of MAIT TCR engagement of MR1 presenting an Escherichia coli-derived stimulatory ligand, rRL-6-CH2OH, previously found in Salmonella typhimurium. We show a clear enhancement of MAIT TCR binding to MR1 due to the presentation of this ligand. Our structure of a MAIT TCR/MR1/rRL-6-CH2OH complex shows an evolutionarily conserved binding orientation, with a clear role for both the CDR3α and CDR3β loops in recognizing the rRL-6-CH2OH stimulatory ligand. We also present two additional xenoreactive MAIT TCR/MR1 complexes that recapitulate the docking orientation documented previously, despite having variation in the CDR2β and CDR3β loop sequences. Our data support a model by which MAIT TCRs engage MR1 in a conserved fashion, with their binding affinities modulated by the nature of the MR1-presented Ag or diversity introduced by alternate Vβ usage or CDR3β sequences.
Temperate phages are pervasive in bacterial genomes, existing as vertically inherited islands termed prophages. Prophages are vulnerable to predation of their host bacterium by exogenous phages. ...Here, we identify BstA, a family of prophage-encoded phage-defense proteins in diverse Gram-negative bacteria. BstA localizes to sites of exogenous phage DNA replication and mediates abortive infection, suppressing the competing phage epidemic. During lytic replication, the BstA-encoding prophage is not itself inhibited by BstA due to self-immunity conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella, and Escherichia prophages is generally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, preventing exploitation by variant BstA-encoding phages. The BstA protein allows prophages to defend host cells against exogenous phage attack without sacrificing the ability to replicate lytically.
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•BstA is an abortive infection protein found in prophages of Gram-negative bacteria•aba, a short DNA sequence within the bstA locus, acts as a self-immunity element•aba gives BstA-encoding prophages immunity to BstA-driven abortive infection•Variant BstA proteins have distinct and cognate aba elements
Prophages can encode abortive infection proteins that give their bacterial hosts population-level phage defense. Owen et al. show that some of these proteins contain internal self-immunity systems so that the prophage is not self-targeted by its own phage-defense protein.
Evidence is mounting that the major histocompatibility complex (MHC) molecule HLA-F (human leukocyte antigen F) regulates the immune system in pregnancy, infection, and autoimmunity by signaling ...through NK cell receptors (NKRs). We present structural, biochemical, and evolutionary analyses demonstrating that HLA-F presents peptides of unconventional length dictated by a newly arisen mutation (R62W) that has produced an open-ended groove accommodating particularly long peptides. Compared to empty HLA-F open conformers (OCs), HLA-F tetramers bound with human-derived peptides differentially stained leukocytes, suggesting peptide-dependent engagement. Our in vitro studies confirm that NKRs differentiate between peptide-bound and peptide-free HLA-F. The complex structure of peptide-loaded β2m-HLA-F bound to the inhibitory LIR1 revealed similarities to high-affinity recognition of the viral MHC-I mimic UL18 and a docking strategy that relies on contacts with HLA-F as well as β2m, thus precluding binding to HLA-F OCs. These findings provide a biochemical framework to understand how HLA-F could regulate immunity via interactions with NKRs.
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•The crystal structure of HLA-F reveals a unique mode of peptide presentation•LIR1 recognizes β2m-HLA-F via a docking strategy that precludes HLA-F OC recognition•Peptide-bound HLA-F and empty HLA-F OCs are recognized by distinct NKRs•Peptide binding increases the proportion of leukocytes that stain with HLA-F tetramer
HLA-F can regulate immunity as an empty open conformer but whether or not HLA-F can present peptides is controversial. Dulberger et al. show that HLA-F has recently evolved an open-ended antigen-binding groove that facilitates presentation of uncharacteristically long peptides and that recognition of HLA-F by NKRs is tunable by peptide binding.
(
) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of
infections is difficult because the bacterium is intrinsically resistant to many ...classes of antibiotics. Developing new and effective treatments against
requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in
by conducting transposon sequencing (TnSeq) on the reference
strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in
, we further characterized
, a predicted penicillin-binding protein and hypothetical lipoprotein (PBP-lipo) that is essential in
and non-essential in
(
). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of
PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the
model organism,
, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11
clinical isolates to several classes of antibiotics, including the β-lactams, ampicillin, and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study
-specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat
infections.
(Mab) is an emerging pathogen that is highly tolerant to current antibiotic therapies, and the current standard of care has a high failure rate. Mycobacteriophages represent a promising alternative ...treatment that have the potential to kill Mab with few side effects. However, the repertoire of phages that infect Mab is limited, and little is understood about the determinants of phage susceptibility in mycobacteria. Two studies from the Hatfull group (R. M. Dedrick, B. E. Smith, R. A. Garlena, D. A. Russell, et al., mBio 12:e03431-20, 2021, https://doi.org/10.1128/mBio.03431-20, and R. M. Dedrick, H. G. Aull, D. Jacobs-Sera, R. A. Garlena, et al., mBio 12:e03441-20, 2021, https://doi.org/10.1128/mBio.03441-20) shed new light on the natural phage complement of Mab and provide some of the first insights into what factors might drive susceptibility to these phages. These studies not only lay the groundwork for therapeutic development of more effective phage therapy in Mab but also provide a foothold for studying how mobile elements such as phages and plasmids impact Mab biology and evolution.
Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αβ T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT ...cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 α1 and α2 helices, similar to classical αβ TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the α-chain, focused on the MR1 α2 helix. TCR β-chain contacts are mostly through the variable CDR3β loop that is positioned proximal to the CDR3α loop directly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of αβ T-cell recognition of MHC and MHC-like ligands.
The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229
(
)clinical isolates has identified an essential ...transcriptional regulator,
, herein called
for resilience regulator, as a frequent target of positive (adaptive) selection.
mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable
to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of
. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.
The genus Mycobacterium includes species such as Mycobacterium tuberculosis, which can cause deadly human diseases. These bacteria have a protective cell envelope that can be remodeled to facilitate ...their survival in challenging conditions. Understanding how such conditions affect membrane remodeling can facilitate antibiotic discovery and treatment. To this end, we describe an optimized fluorogenic probe, N-QTF, that reports on mycolyltransferase activity, which is vital for cell division and remodeling. N-QTF is a glycolipid probe that can reveal dynamic changes in the mycobacterial cell envelope in both fast- and slow-growing mycobacterial species. Using this probe to monitor the consequences of antibiotic treatment uncovered distinct cellular phenotypes. Even antibiotics that do not directly inhibit cell envelope biosynthesis cause conspicuous phenotypes. For instance, mycobacteria exposed to the RNA polymerase inhibitor rifampicin release fluorescent extracellular vesicles (EVs). While all mycobacteria release EVs, fluorescent EVs were detected only in the presence of RIF, indicating that exposure to the drug alters EV content. Macrophages exposed to the EVs derived from RIF-treated cells released lower levels of cytokines, suggesting the EVs moderate immune responses. These data suggest that antibiotics can alter EV content to impact immunity. Our ability to see such changes in EV constituents directly results from exploiting these chemical probes.