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Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

ABSTRACT: S. aureus USA300 isolates utilize the copBL and copAZ gene products to prevent Cu intoxication. We created and examined a ΔcopAZ ΔcopBL mutant strain (cop-). The cop- strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in the cop- background and isolated strains with Tn insertions in the mntABC operon that permitted growth in the presence of Cu. The mutations were in mntA and they were recessive. Under the growth conditions utilized, MntABC functioned in manganese (Mn) import. When cultured with Cu, strains containing a mntA::Tn accumulated less Cu than the parent strain. Mn(II) supplementation improved growth when cop- was cultured with Cu and this phenotype was dependent upon the presence of MntR, which is a repressor of mntABC transcription. A ΔmntR strain had an increased Cu load and decreased growth in the presence of Cu, which was abrogated by the introduction of mntA::Tn. Over-expression of mntABC increased cellular Cu load and sensitivity to Cu. The presence of a mntA::Tn mutation protected iron-sulfur (FeS) enzymes from inactivation by Cu. The data presented are consistent with a model wherein defective MntABC results in decreased cellular Cu accumulation and protection to FeS enzymes from Cu poisoning.

SUBMITTER: Al-Tameemi H

PROVIDER: S-EPMC8121185 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

Al-Tameemi Hassan H Beavers William N WN Norambuena Javiera J Skaar Eric P EP Boyd Jeffrey M JM

Molecular microbiology 20201025 4

S. aureus USA300 isolates utilize the copBL and copAZ gene products to prevent Cu intoxication. We created and examined a ΔcopAZ ΔcopBL mutant strain (cop-). The cop- strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in the cop- background and isolated strains with Tn insertions in the mntABC operon that permitted growth in the presence of Cu. The mutations were in mntA and they were recessive. Under the growth conditions utilized, MntABC f ...[more]

PMID: 33034093

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Project description:Staphylococcus aureus is a human pathogen that has developed several approaches to evade the immune system, including a strategy to resist oxidative killing by phagocytes. This resistance is mediated by production of superoxide dismutase (SOD) enzymes which use manganese as a cofactor. S. aureus encodes two manganese ion transporters, MntABC and MntH, and a possible Nramp family manganese transporter, exemplified by S. aureus N315 SA1432. Their relative contributions to manganese transport have not been well defined in clinically relevant isolates. For this purpose, insertional inactivation mutations were introduced into mntC, mntH, and SA1432 individually and in combination. mntC was necessary for full resistance to methyl viologen, a compound that generates intracellular free radicals. In contrast, strains with an intact mntH gene had a minimal increase in resistance that was revealed only in mntC strains, and no change was observed upon mutation of SA1432 in strains lacking both mntC and mntH Similarly, MntC alone was required for high cellular SOD activity. In addition, mntC strains were attenuated in a murine sepsis model. To further link these observations to manganese transport, an S. aureus MntC protein lacking manganese binding activity was designed, expressed, and purified. While circular dichroism experiments demonstrated that the secondary and tertiary structures of this protein were unaltered, a defect in manganese binding was confirmed by isothermal titration calorimetry. Unlike complementation with wild-type mntC, introduction of the manganese-binding defective allele into the chromosome of an mntC strain did not restore resistance to oxidative stress or virulence. Collectively, these results underscore the importance of MntC-dependent manganese transport in S. aureus oxidative stress resistance and virulence.IMPORTANCE Work outlined in this report demonstrated that MntC-dependent manganese transport is required for S. aureus virulence. These study results support the model that MntC-specific antibodies elicited by a vaccine have the potential to disrupt S. aureus manganese transport and thus abrogate to its virulence.

Project description:Urinary tract infection (UTI) is one of the most common infectious conditions affecting people in the United States and around the world. Our knowledge of the host-pathogen interaction during UTI caused by Gram-positive bacterial uropathogens is limited compared to that for Gram-negative pathogens. Here, we investigated whether copper and the primary copper-containing protein, ceruloplasmin, are mobilized to urine during naturally occurring UTI caused by Gram-positive uropathogens in patients. Next, we probed the role of copper resistance in the fitness of methicillin-resistant Staphylococcus aureus (MRSA) during experimental UTI in a murine model. Our findings demonstrate that urinary copper and ceruloplasmin content are elevated during UTI caused by Enterococcus faecalis, S. aureus, S. epidermidis, and S. saprophyticus. MRSA strains successfully colonize the urinary tract of female CBA mice with selective induction of inflammation in the kidneys but not the bladder. MRSA mutants lacking CopL, a copper-binding cell surface lipoprotein, and the ACME genomic region containing copL, exhibit decreased fitness in the mouse urinary tract compared to parental strains. Copper sensitivity assays, cell-associated copper and iron content, and bioavailability of iron during copper stress demonstrate that homeostasis of copper and iron is interlinked in S. aureus. Importantly, relative fitness of the MRSA mutant lacking the ACME region is further decreased in mice that receive supplemental copper compared to the parental strain. In summary, copper is mobilized to the urinary tract during UTI caused by Gram-positive pathogens, and copper resistance is a fitness factor for MRSA during UTI. IMPORTANCE Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics. Better understanding of the host-pathogen interface is critical for development of novel interventional strategies. Here, we sought to elucidate the role of copper in host-Staphylococcus aureus interaction during UTI. Our results reveal that copper is mobilized to the urine as a host response in patients with UTI. Our findings from the murine model of UTI demonstrate that copper resistance is involved in the fitness of methicillin-resistant S. aureus (MRSA) during interaction with the host. We also establish a critical link between adaptation to copper stress and iron homeostasis in S. aureus.

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:Acquisition of numerous virulence determinants affords Staphylococcus aureus greater pathogenicity than other skin-colonizing staphylococci in humans. Additionally, the metabolic adaptation of S. aureus to nonrespiratory conditions encountered during infection (e.g., hypoxia, nitric oxide, iron chelation) has been implicated as contributing to S. aureus virulence. Specifically, S. aureus has been shown to ferment glycolytic substrates in nonrespiratory environments encountered within the host. Here, we show that S. aureus has acquired unique carbohydrate transporters that facilitate the maximal uptake of host sugars and serve to support nonrespiratory growth in inflamed tissue. The carbohydrate substrates of 11 S. aureus transporters were identified, and at least four of their genes encode S. aureus glucose transporters (glcA, glcB, glcC, and glcU). Moreover, two transporter genes (glcA and glcC) are unique to S. aureus and contribute disproportionately to the nonrespiratory growth of S. aureus on glucose. Targeted inactivation of sugar transporters reduced glucose uptake and attenuated S. aureus in a murine model of skin and soft tissue infections. These data expand the evidence for metabolic adaptation of S. aureus to invasive infection and demonstrate the specific requirement for the fermentation of glucose over all other available carbohydrates. Ultimately, acquisition of foreign genes allows S. aureus to adopt a metabolic strategy resembling that of infiltrating host immune cells: high glycolytic flux coupled to lactate excretion. The bacterial pathogen Staphylococcus aureus causes a wide range of human infections that are costly and difficult to treat. S. aureus differs from closely related commensal staphylococci in its ability to flourish following the invasion of deeper tissue from the skin surface. There, S. aureus primarily uses glucose to grow under respiration-limiting conditions imposed by the immune system. It was previously unclear how S. aureus thrives in this environment when other Staphylococcus species cannot. Our results provide evidence that S. aureus has acquired an expanded repertoire of carbohydrate transporters. In particular, four glucose transporters contribute to efficient S. aureus growth during infection. Thus, S. aureus has evolved to maximize its glucose uptake abilities for enhanced glycolytic flux during tissue invasion. This dependence on glucose acquisition for S. aureus virulence may also explain links between serious infectious complications associated with diabetic patients exhibiting elevated blood glucose levels.

Dataset Information

Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

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Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

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