Jason W. Rosch

Streptococcus pneumoniae is a leading human respiratory pathogen, yet its success depends not on disease but on its ability to persist as an asymptomatic colonizer of the nasopharynx. The nasopharyngeal mucosa is lined with mucus, whose heavily O-glycosylated mucin proteins are the most abundant macromolecules in the pneumococcal natural niche. While mucus is canonically viewed as a host defense barrier, we found that mucin actively reprograms pneumococcal physiology to promote colonization through two distinct mechanisms. First, mucin suppresses pneumococcal autolysis by specifically inhibiting the enzymatic activity of the major autolysin LytA, prolonging bacterial survival and restricting release of the cholesterol-dependent cytotoxin pneumolysin, thereby reducing host cell damage. Second, mucin induces de novo biosynthesis and extracellular release of tryptophan, an essential amino acid that humans cannot synthesize and that serves as a substrate for host immunomodulatory pathways. Tryptophan biosynthesis was critical for pneumococcal survival against macrophage killing, and its disruption abolished both colonization and invasive disease. Transcriptomic analysis revealed that tryptophan biosynthesis enables the pneumococcus to survive a bactericidal macrophage state while preventing macrophage transition to proliferative tissue repair. Together, these findings reveal that mucin is not merely a barrier but an active signal that reprograms the pneumococcus toward a persistent, host-tolerant state, identifying a distinctive repertoire of colonization mechanisms that allow a virulent respiratory pathogen to sustain long-term asymptomatic carriage in its human reservoir.

Streptococcus pneumoniae rapidly translocates across polarized human bronchial epithelial barriers, with viable bacteria recovered from the basolateral compartment within 1 h post-infection. Disruption of the pyruvate node through combined deletion of pyruvate oxidase ( spxB ) and lactate oxidase ( lctO ) markedly enhanced transmigration of S. pneumoniae across polarized Calu-3 monolayers without causing early cytotoxicity or loss of monolayer integrity. This hyper-invasive phenotype was conserved in the TIGR4 and EF3030 background and under air-liquid interface conditions. Importantly, single Δ lctO mutants exhibited significantly greater translocation than Δ spxB mutants or wild-type strains across bronchial (Calu-3), alveolar (A549), and pharyngeal (Detroit 562) epithelial models. Enhanced translocation correlated with increased bacterial adherence but was independent of capsule expression, extracellular H₂O₂ production, pneumolysin, or tight junction disruption, as evidenced by stable transepithelial electrical resistance (TEER), lack of caspase-3/7 activation, and minimal IL-18 release at early time points. High-resolution confocal microscopy revealed intracellular Δ lctO pneumococci localized within N-acetylglucosamine/sialic acid (GN/SA)-containing compartments as early as 1 h post-infection. In murine macrophages, Δ lctO mutants were phagocytosed at rates similar to wild-type bacteria but induced greater pneumolysin-dependent cytotoxicity at 24 h. These findings demonstrate that LctO functions as a metabolic checkpoint that restrains pneumococcal invasion of respiratory epithelia, revealing a previously unrecognized role for lactate oxidase in controlling the transition from colonization to invasive disease. Importance: This study identifies lactate oxidase (LctO) as a critical metabolic checkpoint that restrains Streptococcus pneumoniae invasion of respiratory epithelial barriers. By linking pyruvate node metabolism to the control of transmigration, these findings reveal a novel mechanism by which central carbon metabolism regulates pneumococcal virulence and the transition from colonization to invasive disease.

Bacteria can survive antibiotic treatment through phenotypic adaptation, a process that is often transient and involves heterogeneous transcriptional reprogramming. However, how this transcriptional heterogeneity is generated and how it influences antibiotic survival remains unclear. Here, we use bacterial single-cell RNA sequencing and functional assays in Klebsiella pneumoniae to characterize transcriptional heterogeneity and examine how pre-treatment cell states are associated with antibiotic-induced responses and survival outcomes. Using growth phase as a biologically meaningful axis of transcriptional variation, we reveal that even within an isogenic population, distinct transcriptional responses can be induced and co-contribute to survival. These responses are shaped by the cell's pre-treatment transcriptional state and the mechanism of antibiotic action. Genetic and environmental perturbations, such as rpoS deletion and nutrient supplementation, shift pre-treatment cell states and alter survival frequencies. Our findings establish the biological significance of transcriptional heterogeneity shaped by pre-treatment cell states, providing a systems-level framework for understanding bacterial antibiotic response and suggesting strategies to enhance antibiotic efficacy by modulating cell states.

SUMMARY: The mechanisms underlying adaptation to antibiotic pressure within complex host environments remain incompletely understood. By experimentally evolving Streptococcus pneumoniae subjected to various antibiotics and immune states, we demonstrate populations adopting distinct adaptive strategies depending on specific selective context. General antibiotic stress drives convergent mutations in rny , encoding the RNA degradosome scaffold RNase Y, that exhibit broad-spectrum antibiotic tolerance and accelerated recovery. Single-cell transcriptomics revealed antibiotic-induced death is driven by transcriptional collapse, a catastrophic loss of RNA quantity and integrity. In contrast, rny mutants avert this via a bet-hedging strategy: a resilient minority maintains a baseline transcriptional profile, while a quiescent majority undergoes selective RNA degradation to preserve transcript fidelity. Upon stress removal, these populations execute a prioritized transcriptional ribosomal reboot, facilitating accelerated recovery. These findings identify RNA turnover as a tunable master regulator to survive combined pressures of antibiotics and immunity.

Acinetobacter baumannii is an Enterococcus , Staphylococcus , Klebsiella , Acinetobacter , Pseudomonas , and Enterobacter species (ESKAPE) pathogen linked to healthcare-associated diseases. Due to evolved resistance, last-resort antibiotics such as the lipooligosaccharide (LOS)-targeting colistin are increasingly used to treat multidrug-resistant isolates. To track the evolution of colistin resistance within a host, we performed sequential oropharyngeal infections in immunocompetent or immune-depleted mice in the presence of inhaled colistin. Both resistant and heteroresistant A. baumannii strains emerged with pmrB mutations that efficiently competed with the susceptible parent in the presence of colistin. These pmrB mutants had a fitness cost in the absence of colistin treatment but retained their ability to colonize the host. In contrast, LOS-deficient A. baumannii mutants removed the target of colistin, but such mutants were unable to colonize the lung. The two pathogenic pmrB mutants showed clear evidence of LOS modification, which was linked to increased transcription of LOS modification enzymes, including the product of the cryptic eptA gene. Spontaneous insertion mutations that caused hyperexpression of eptA allowed the heteroresistant mutant to develop clinically significant colistin resistance. Insertion mutations upstream of the eptA gene or those disrupting hns , which encodes a small histone-like protein, resulted in increased eptA transcript, linking expression of this protein to clinically significant resistance. A resistant variant derived from the heteroresistant parent was stable in the absence of drug, but continued passaging selected for colistin-resensitized pseudorevertants that were largely due to disruption of the LOS modification enzymes. Therefore, colistin heteroresistance is an early stage in the stepwise acquisition of stable colistin resistance in A. baumannii .

Synergy between influenza A virus (IAV) and Streptococcus pneumoniae is a long-recognized and clinically important problem. Recent work has demonstrated that IAV particles can directly bind to the bacterial surface and that bacterial-viral complexes exhibit enhanced bacterial colonization and invasive disease, increased viral environmental survival leading to increased efficacy of airborne transmission, and enhanced vaccine response to both pathogens over simultaneous co-infection without direct interactions. However, the molecule(s) responsible for mediating the direct interaction are yet to be characterized. In this study, we demonstrate that the broadly conserved Gram-positive bacterial cell wall glycan lipoteichoic acid (LTA) is one of the molecules that can mediate this interaction. This interaction between viral particles and bacterial cell-envelope glycans is also demonstrated in interactions between enteric viruses and enteric bacteria, suggesting a conserved mechanism of trans-kingdom interactions. We show that LTA will compete for binding between IAV and S. pneumoniae , that disruption of genes responsible for LTA presentation at the cell surface will reduce viral binding, and that viral neuraminidase can bind LTA. This work adds to the growing body of literature on direct bacterial-viral interactions between human-associated bacteria and pathogenic viruses and can provide novel insights into the lethal synergy of influenza-pneumococcal co-infections. IMPORTANCE: Co-infection between influenza A virus (IAV) and Streptococcus pneumoniae leads to severe disease. Recently, it was demonstrated that IAV particles can bind to the surface of bacterial cells and that direct interactions will enhance both bacterial and viral pathogenesis as well as immune responses to each pathogen. However, it is unclear what bacterial and viral components are responsible for the interaction. We demonstrate that a carbohydrate component of the bacterial cell wall can bind to IAV particles. This is similar to direct interactions observed between enteric viruses and cell wall components of enteric bacteria. This work adds to the body of knowledge about trans-kingdom interactions between human-associated bacteria and human pathogenic viruses, as well as providing novel insights into the serious clinical problem of influenza-pneumococcal synergy.

Non-tuberculous mycobacteria are emerging pathogens with high intrinsic drug resistance. Among these, Mycobacterium abscessus is particularly refractory owing to its extensive array of resistance mechanisms. Here we introduce florfenicol amine (FF-NH ₂ ), a major metabolite of the antibiotic florfenicol, which acts as a prodrug with narrow-spectrum activity against M. abscessus−chelonae complex species. FF-NH ₂ leverages intrinsic M. abscessus resistance conferred by the transcription factor WhiB7. It avoids WhiB7-dependent resistance mediated by the O -acetyltransferase Cat and is activated by the WhiB7-dependent N -acetyltransferase Eis2 in a prodrug fashion to generate the active translational inhibitor FF acetyl (FF-ac). FF-NH ₂ induces Eis2 expression through WhiB7, creating a feed-forward bioactivation loop, which increases FF-ac accumulation and antimicrobial action. FF-NH ₂ displays antiresistance properties, can synergize with other antibiotics and mitigates toxicity linked to mammalian mitochondrial ribosome inhibition. Importantly, FF-NH ₂ demonstrated efficacy in a murine model of M. abscessus infection. These findings suggest intrinsic resistance can be exploited to develop safer and more effective treatments for this pathogen.

Profiles of human nasal colonization consistently demonstrate that Staphylococcus aureus and Streptococcus pneumoniae can co-exist in the nasopharynx. Several studies have demonstrated the antagonist relationship between the two organisms via several molecular mechanisms, including competition for nutrients as well as via direct killing by hydrogen peroxide. During nasal colonization, the pneumococcus is in direct contact with the fatty acid h 18:0, which is released into the extracellular environment by S. aureus . We report that h 18:0 is specifically toxic to the pneumococcus among the pathogenic streptococci, providing a unique mechanism for interspecies competition during colonization. Exposure of cells to h 18:0 revealed that S. pneumoniae could rapidly adapt to and overcome the observed toxicity. Whole-genome analysis revealed the mechanism underlying this resistance being linked to a truncation of a glycosyltransferase in the capsule biosynthesis locus and a genomic inversion in the phase variation locus, leading to altered cell surface charge and membrane lipid composition. These physiological differences in the resistant isolates may aid in repelling toxic, charged fatty acids such as h 18:0 from the cell membrane. IMPORTANCE: The pneumococcus and S. aureus are two of the most well-characterized residents of the human nasopharynx; yet much remains unknown regarding how the two bacteria interact. Here, we describe the potential of S. aureus -produced h 18:0, whose function and biological impact are still being described, to act as an interspecies competition molecule against S. pneumoniae , and how the pneumococcus can adapt to overcome its toxicity.

Acinetobacter baumannii is an ESKAPE pathogen linked to healthcare-associated diseases. Due to evolved resistance, last-resort antibiotics such as the lipooligosaccharide (LOS)-targeting colistin are increasingly used to treat multidrug-resistant isolates. To track the evolution of colistin resistance within a host, we performed sequential oropharyngeal infections in immunocompetent or immune-depleted mice in the presence of inhaled colistin. Both resistant and heteroresistant A. baumannii strains emerged with pmrB mutations that efficiently competed with the susceptible parent in the presence of colistin. These pmrB mutants had a fitness cost in the absence of colistin treatment but retained their ability to colonize the host. In contrast, LOS-deficient A. baumannii mutants removed the target of colistin, but such mutants were unable to colonize the lung. The two pathogenic pmrB mutants showed clear evidence of LOS modification, which was linked to increased transcription of LOS modification enzymes, including the product of the cryptic eptA gene. Spontaneous insertion mutations that caused hyperexpression of eptA allowed the heteroresistant mutant to develop clinically-significant colistin resistance. Insertion mutations upstream of the eptA gene or those disrupting hns , which encodes a small histone-like protein, resulted in increased eptA transcript, linking expression of this protein to clinically significant resistance. A resistant variant derived from the heteroresistant parent was stable in the absence of drug, but continued passaging selected for colistin-resensitized pseudorevertants that were largely due to disruption of the LOS modification enzymes. Therefore, colistin heteroresistance is an early stage in the stepwise acquisition of stable colistin resistance in A. baumannii . Significance: The mutational pathways leading to antibiotic-resistant infections and the role of the immune system in preventing them are poorly understood. Here we employed a colistin-treated mouse pneumonia model of Acinetobacter baumannii and observed the evolution of colistin-resistant and heteroresistant mutants in immune-depleted and immunocompetent hosts, respectively. We show that mutations that result in alteration of colistin’s target drive evolutionary pathways to resistance, whereas removal of the target is unlikely to be clinically significant. We demonstrated that the heteroresistant mutant generates subpopulations with higher levels of resistance through insertion mutations in specific gene regions. This study furthers our understanding of how resistance emerges during infection and provides a genetic explanation for the transition from colistin heteroresistance to full resistance.