The redox-responsive transcriptional regulator Rex represses fermentative metabolism and is required for Listeria monocytogenes pathogenesis.

The Gram-positive bacterium Listeria monocytogenes is the causative agent of the foodborne disease listeriosis, one of the deadliest bacterial infections known. In order to cause disease, L. monocytogenes must properly coordinate its metabolic and virulence programs in response to rapidly changing environments within the host. However, the mechanisms by which L. monocytogenes senses and adapts to the many stressors encountered as it transits through the gastrointestinal (GI) tract and disseminates to peripheral organs are not well understood. In this study, we investigated the role of the redox-responsive transcriptional regulator Rex in L. monocytogenes growth and pathogenesis. Rex is a conserved canonical transcriptional repressor that monitors the intracellular redox state of the cell by sensing the ratio of reduced and oxidized nicotinamide adenine dinucleotides (NADH and NAD+, respectively). Here, we demonstrated that L. monocytogenes Rex represses fermentative metabolism and is therefore required for optimal growth in the presence of oxygen. We also show that in vitro, Rex represses the production of virulence factors required for survival and invasion of the GI tract, as a strain lacking rex was more resistant to acidified bile and invaded host cells better than wild type. Consistent with these results, Rex was dispensable for colonizing the GI tract and disseminating to peripheral organs in an oral listeriosis model of infection. However, Rex-dependent regulation was required for colonizing the spleen and liver, and L. monocytogenes lacking the Rex repressor were nearly sterilized from the gallbladder. Taken together, these results demonstrated that Rex functions as a repressor of fermentative metabolism and suggests a role for Rex-dependent regulation in L. monocytogenes pathogenesis. Importantly, the gallbladder is the bacterial reservoir during listeriosis, and our data suggest redox sensing and Rex-dependent regulation are necessary for bacterial survival and replication in this organ.

Listeria monocytogenes SpxA1 is a global regulator required to activate genes encoding catalase and heme biosynthesis enzymes for aerobic growth.

An imbalance of cellular oxidants and reductants causes redox stress, which must be rapidly detected to restore homeostasis. In bacteria, the Firmicutes encode conserved Spx-family transcriptional regulators that modulate transcription in response to redox stress. SpxA1 is an Spx-family orthologue in the intracellular pathogen Listeria monocytogenes that is essential for aerobic growth and pathogenesis. Here, we investigated the role of SpxA1 in growth and virulence by identifying genes regulated by SpxA1 in broth and during macrophage infection. We found SpxA1-activated genes encoding heme biosynthesis enzymes and catalase (kat) were required for L. monocytogenes aerobic growth in rich medium. An Spx-recognition motif previously defined in Bacillus subtilis was identified in the promoters of SpxA1-activated genes and proved necessary for the proper activation of two genes, indicating this regulation by SpxA1 is likely direct. Together, these findings elucidated the mechanism of spxA1 essentiality in vitro and demonstrated that SpxA1 is required for basal expression of scavenging enzymes to combat redox stress generated in the presence of oxygen.

Catabolic Ornithine Carbamoyltransferase Activity Facilitates Growth of Staphylococcus aureus in Defined Medium Lacking Glucose and Arginine.

Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host.

Investigating the Roles of Listeria monocytogenes Peroxidases in Growth and Virulence.

Bacteria have necessarily evolved a protective arsenal of proteins to contend with peroxides and other reactive oxygen species generated in aerobic environments. Listeria monocytogenes encounters an onslaught of peroxide both in the environment and during infection of the mammalian host, where it is the causative agent of the foodborne illness listeriosis. Despite the importance of peroxide for the immune response to bacterial infection, the strategy by which L. monocytogenes protects against peroxide toxicity has yet to be illuminated. Here, we investigated the expression and essentiality of all the peroxidase-encoding genes during L. monocytogenes growth in vitro and during infection of murine cells in tissue culture. We found that chdC and kat were required for aerobic growth in vitro, and fri and ahpA were each required for L. monocytogenes to survive acute peroxide stress. Despite increased expression of fri, ahpA, and kat during infection of macrophages, only fri proved necessary for cytosolic growth. In contrast, the proteins encoded by lmo0367, lmo0983, tpx, lmo1609, and ohrA were dispensable for aerobic growth, acute peroxide detoxification, and infection. Together, our results provide insight into the multifaceted L. monocytogenes peroxide detoxification strategy and demonstrate that L. monocytogenes encodes a functionally diverse set of peroxidase enzymes. IMPORTANCE Listeria monocytogenes is a facultative intracellular pathogen and the causative agent of the foodborne illness listeriosis. L. monocytogenes must contend with reactive oxygen species generated extracellularly during aerobic growth and intracellularly by the host immune system. However, the mechanisms by which L. monocytogenes defends against peroxide toxicity have not yet been defined. Here, we investigated the roles of each of the peroxidase-encoding genes in L. monocytogenes growth, peroxide stress response, and virulence in mammalian cells.

Amino Acid Catabolism in Staphylococcus aureus and the Function of Carbon Catabolite Repression.

Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kinase, also abrogated growth of JE2 in CDM, suggesting that ATP production from pyruvate-producing amino acids is also critical for growth. In addition, although a functional respiratory chain was absolutely required for growth, the oxygen consumption rate and intracellular ATP concentration were significantly lower during growth in CDM than during growth in glucose-containing media. Finally, transcriptional analyses demonstrated that expression levels of genes coding for the enzymes that synthesize glutamate from proline, arginine, and histidine are repressed by CcpA and carbon catabolite repression. These data show that pathways important for glutamate catabolism or ATP generation via Pta/AckA are important for growth in niches where glucose is not abundant, such as abscesses within skin and soft tissue infections.IMPORTANCES. aureus is a significant cause of both morbidity and mortality worldwide. This bacterium causes infections in a wide variety of organ systems, the most common being skin and soft tissue. Within a staphylococcal abscess, levels of glucose, a preferred carbon source, are limited due to the host immune response. Therefore, S. aureus must utilize other available carbon sources such as amino acids or peptides to proliferate. Our results show that glutamate and amino acids that serve as substrates for glutamate synthesis, particularly proline, function as major carbon sources during growth, whereas other amino acids that generate pyruvate are important for ATP synthesis via substrate-level phosphorylation in the Pta-AckA pathway. Our data support a model whereby certain amino acid catabolic pathways, and acquisition of those particular amino acids, are crucial for growth in niches where glucose is not abundant.

Conjugative Transfer in Staphylococcus aureus.

The acquisition of plasmids has led to a significant increase in antimicrobial resistance within the staphylococci. In order to study these plasmids effectively, one must be able move the plasmid DNA into genetically clean backgrounds. While the smaller staphylococcal class I (1-5 kb) and class II (10-30 kb) plasmids are readily transferred using bacteriophage transduction or electroporation, these methods are inefficient at moving the larger class III (30-60 kb) plasmids. This review describes methods to transfer class III plasmids via conjugative mobilization.