CanB is a metabolic mediator of antibiotic resistance in Neisseria gonorrhoeae.

The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments and antibiotics. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations. We hypothesized that the requirement for supplemental CO2, present in approximately half of isolates, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO2 dependence is attributable to a single substitution in a ß-carbonic anhydrase, CanB. CanB19E is necessary and sufficient for growth in the absence of CO2, and the hypomorphic CanB19G variant confers CO2 dependence. Furthermore, ciprofloxacin resistance is correlated with CanB19G in clinical isolates, and the presence of CanB19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has affected the acquisition of fluoroquinolone resistance.

Nontoxigenic Vibrio cholerae Challenge Strains for Evaluating Vaccine Efficacy and Inferring Mechanisms of Protection.

Human challenge studies are instrumental for testing cholera vaccines, but these studies use outdated strains and require inpatient facilities. Here, we created next-generation isogenic Ogawa and Inaba O1 V. cholerae challenge strains (ZChol strains) derived from a contemporary Zambian clinical isolate representative of current dominant pandemic V. cholerae. Since the primary mechanism of immune protection against cholera is thought to be antibody responses that limit V. cholerae colonization and not the diarrheagenic actions of cholera toxin, these strains were rendered nontoxigenic. In infant mice, the ZChol strains did not cause diarrhea and proved to accurately gauge reduction in intestinal colonization mediated by effective vaccination. ZChol strains were also valuable as targets for measuring vibriocidal antibody responses. Using barcoded ZChol strains, we discovered that vaccination and passive immunity in the infant mouse model tightens the infection bottleneck without restricting pathogen expansion during intestinal infection. Collectively, our findings suggest that ZChol strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera. IMPORTANCE Human challenge studies are a valuable method for testing the efficacy of cholera vaccines. However, challenge studies cannot be performed in countries of cholera endemicity due to safety concerns; also, contemporary pandemic Vibrio cholerae strains are not used in current challenge studies. To facilitate cholera research, we derived nontoxigenic challenge strains of both V. cholerae serotypes from a 2016 clinical isolate from Zambia and demonstrated how they can be used to gauge cholera immunity accurately and safely. These strains were also genetically barcoded, adding the potential for analyses of V. cholerae population dynamics to challenge studies. Preclinical analyses presented here suggest that these strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera.

Adaptation to the cervical environment is associated with increased antibiotic susceptibility in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.

The frontiers of addressing antibiotic resistance in Neisseria gonorrhoeae.

The sexually transmitted infection gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae, can cause urethritis, cervicitis, and systemic disease, among other manifestations. N. gonorrhoeae has rapidly rising incidence along with increasing levels of antibiotic resistance to a broad range of drugs including first-line treatments. The rise in resistance has led to fears of untreatable gonorrhea causing substantial disease globally. In this review, we will describe multiple approaches being undertaken to slow and control this spread of resistance. First, a number of old drugs have been repurposed and new drugs are being developed with activity against Neisseria gonorrhoeae. Second, vaccine development, long an important goal, is advancing. Third, new diagnostics promise rapid detection of antibiotic resistance and a shift from empiric to tailored treatment. The deployment of these new tools for addressing the challenge of antibiotic resistance will require careful consideration to provide optimal care for all patients while extending the lifespan of treatment regimens.

A Compendium of Genetic Modifiers of Mitochondrial Dysfunction Reveals Intra-organelle Buffering.

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.

Genomics and proteomics of mycobacteriophage patience, an accidental tourist in the Mycobacterium neighborhood.

UNLABELLED: Newly emerging human viruses such as Ebola virus, severe acute respiratory syndrome (SARS) virus, and HIV likely originate within an extant population of viruses in nonhuman hosts and acquire the ability to infect and cause disease in humans. Although several mechanisms preventing viral infection of particular hosts have been described, the mechanisms and constraints on viral host expansion are ill defined. We describe here mycobacteriophage Patience, a newly isolated phage recovered using Mycobacterium smegmatis mc(2)155 as a host. Patience has genomic features distinct from its M. smegmatis host, including a much lower GC content (50.3% versus 67.4%) and an abundance of codons that are rarely used in M. smegmatis. Nonetheless, it propagates well in M. smegmatis, and we demonstrate the use of mass spectrometry to show expression of over 75% of the predicted proteins, to identify new genes, to refine the genome annotation, and to estimate protein abundance. We propose that Patience evolved primarily among lower-GC hosts and that the disparities between its genomic profile and that of M. smegmatis presented only a minimal barrier to host expansion. Rapid adaptions to its new host include recent acquisition of higher-GC genes, expression of out-of-frame proteins within predicted genes, and codon selection among highly expressed genes toward the translational apparatus of its new host. IMPORTANCE: The mycobacteriophage Patience genome has a notably lower GC content (50.3%) than its Mycobacterium smegmatis host (67.4%) and has markedly different codon usage biases. The viral genome has an abundance of codons that are rare in the host and are decoded by wobble tRNA pairing, although the phage grows well and expression of most of the genes is detected by mass spectrometry. Patience thus has the genomic profile of a virus that evolved primarily in one type of host genetic landscape (moderate-GC bacteria) but has found its way into a distinctly different high-GC environment. Although Patience genes are ill matched to the host expression apparatus, this is of little functional consequence and has not evidently imposed a barrier to migration across the microbial landscape. Interestingly, comparison of expression levels and codon usage profiles reveals evidence of codon selection as the genome evolves and adapts to its new environment.

Mycobacterium tuberculosis ClpP1 and ClpP2 function together in protein degradation and are required for viability in vitro and during infection.

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy.