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Growth and Stress Tolerance Comprise Independent Metabolic Strategies Critical for Staphylococcus aureus Infection.

ABSTRACT: Staphylococcus aureus is an important pathogen that leads to high morbidity and mortality. Although S. aureus produces many factors important for pathogenesis, few have been validated as playing a role in the pathogenesis of S. aureus pneumonia. To gain a better understanding of the genetic elements required for S. aureus pathogenesis in the airway, we performed an unbiased genome-wide transposon sequencing (Tn-seq) screen in a model of acute murine pneumonia. We identified 136 genes important for bacterial survival during infection, with a high proportion involved in metabolic processes. Phenotyping 80 individual deletion mutants through diverse in vitro and in vivo assays demonstrated that metabolism is linked to several processes, which include biofilm formation, growth, and resistance to host stressors. We further validated the importance of 23 mutations in pneumonia. Multivariate and principal-component analyses identified two key metabolic mechanisms enabling infection in the airway, growth (e.g., the ability to replicate and form biofilms) and resistance to host stresses. As deep validation of these hypotheses, we investigated the role of pyruvate carboxylase, which was important across multiple infection models and confirmed a connection between growth and resistance to host cell killing. Pathogenesis is conventionally understood in terms of the host-pathogen interactions that enable a pathogen to neutralize a host's immune response. We demonstrate with the important bacterial pathogen S. aureus that microbial metabolism influences key traits important for in vivo infection, independent from host immunomodulation. IMPORTANCE Staphylococcus aureus is an important bacterial pathogen that causes significant morbidity and mortality, infecting numerous bodily sites, including the respiratory tract. To identify the bacterial requirements for lung infection, we conducted a genome-wide screen in a mouse model of acute pneumonia. We discovered that metabolic genes were overrepresented in those required for lung infection. In contrast to the conventional view of pathogenesis focusing on immunomodulation, we demonstrate through phenotyping of deletion mutants in several functional assays that replicative ability and tolerance against host defenses form two key metabolic dimensions of bacterial infection. These dimensions are independent for most pathways but are coupled in central carbon metabolism and highlight the critical role of bacterial metabolism in survival against host defenses during infection.

SUBMITTER: Kim GL

PROVIDER: S-EPMC8262855 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Manganese (Mn) is an essential micronutrient critical for the pathogenesis of Staphylococcus aureus, a significant cause of human morbidity and mortality. Paradoxically, excess Mn is toxic; therefore, maintenance of intracellular Mn homeostasis is required for survival. Here we describe a Mn exporter in S. aureus, MntE, which is a member of the cation diffusion facilitator (CDF) protein family and conserved among Gram-positive pathogens. Upregulation of mntE transcription in response to excess Mn is dependent on the presence of MntR, a transcriptional repressor of the mntABC Mn uptake system. Inactivation of mntE or mntR leads to reduced growth in media supplemented with Mn, demonstrating MntE is required for detoxification of excess Mn. Inactivation of mntE results in elevated levels of intracellular Mn, but reduced intracellular iron (Fe) levels, supporting the hypothesis that MntE functions as a Mn efflux pump and Mn efflux influences Fe homeostasis. Strains inactivated for mntE are more sensitive to the oxidants NaOCl and paraquat, indicating Mn homeostasis is critical for resisting oxidative stress. Furthermore, mntE and mntR are required for full virulence of S. aureus during infection, suggesting S. aureus experiences Mn toxicity in vivo Combined, these data support a model in which MntR controls Mn homeostasis by balancing transcriptional repression of mntABC and induction of mntE, both of which are critical for S. aureus pathogenesis. Thus, Mn efflux contributes to bacterial survival and virulence during infection, establishing MntE as a potential antimicrobial target and expanding our understanding of Mn homeostasis.IMPORTANCE Manganese (Mn) is generally viewed as a critical nutrient that is beneficial to pathogenic bacteria due to its function as an enzymatic cofactor and its capability of acting as an antioxidant; yet paradoxically, high concentrations of this transition metal can be toxic. In this work, we demonstrate Staphylococcus aureus utilizes the cation diffusion facilitator (CDF) family protein MntE to alleviate Mn toxicity through efflux of excess Mn. Inactivation of mntE leads to a significant reduction in S. aureus resistance to oxidative stress and S. aureus-mediated mortality within a mouse model of systemic infection. These results highlight the importance of MntE-mediated Mn detoxification in intracellular Mn homeostasis, resistance to oxidative stress, and S. aureus virulence. Therefore, this establishes MntE as a potential target for development of anti-S. aureus therapeutics.

Project description:Staphylococcus aureus is an opportunistic pathogen with a clinical spectrum ranging from asymptomatic skin colonization to invasive infections. While traditional antibiotic therapies can be effective against S. aureus, the increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. Photodynamic inactivation (PDI) is an innovative and promising alternative to antibiotics. While progress has been made in our understanding of the bacterial response to PDI, major gaps remain in our knowledge of PDI tolerance, the global cellular response, and adaptive genomic mutations acquired as a result of PDI. To address these gaps, S. aureus HG003 and isogenic mutants with mutations in agr, mutS, mutL, and mutY exposed to single or multiple doses of PDI were assessed for survival and tolerance and examined by global transcriptome and genome analyses to identify regulatory and genetic adaptations that contribute to tolerance. Pathways in inorganic ion transport, oxidative response, DNA replication recombination and repair, and cell wall and membrane biogenesis were identified in a global cellular response to PDI. Tolerance to PDI was associated with superoxide dismutase and the S. aureus global methylhydroquinone (MHQ)-quinone transcriptome network. Genome analysis of PDI-tolerant HG003 identified a nonsynonymous mutation in the quinone binding domain of the transcriptional repressor QsrR, which mediates quinone sensing and oxidant response. Acquisition of a heritable QsrR mutation through repeated PDI treatment demonstrates selective adaption of S. aureus to PDI. PDI tolerance of a qsrR gene deletion in HG003 confirmed that QsrR regulates the S. aureus response to PDI.IMPORTANCE Staphylococcus aureus can cause disease at most body sites, with illness ranging from asymptomatic infection to death. The increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. S. aureus acquires resistance to antibiotics through multiple mechanisms, often by genetic variation that alters antimicrobial targets. Photodynamic inactivation (PDI), which employs a combination of a nontoxic dye and low-intensity visible light, is a promising alternative to antibiotics that effectively eradicates S. aureus in human infections when antibiotics are no longer effective. In this study, we demonstrate that repeated exposure to PDI results in resistance of S. aureus to further PDI treatment and identify the underlying bacterial mechanisms that contribute to resistance. This work supports further analysis of these mechanisms and refinement of this novel technology as an adjunctive treatment for S. aureus infections.

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Growth and Stress Tolerance Comprise Independent Metabolic Strategies Critical for Staphylococcus aureus Infection.

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