Journal of Bacteriology最新文献

Staphylococcus aureus and Pseudomonas aeruginosa are the two pathogens that colonize the airway of cystic fibrosis patients. As patients age, P. aeruginosa outcompetes S. aureus to become the predominant organism in the airway, which overlaps with worsening symptoms. This inverse correlation is partly due to the ability of P. aeruginosa to secrete secondary metabolites and virulence factors that are antagonistic to the host cells and other bacteria present. Several of these secondary metabolites inhibit S. aureus respiration. SaeRS is a two-component regulatory system that promotes the transcription of numerous virulence genes in S. aureus. The transcription of SaeRS-regulated genes is decreased as a function of respiratory status. The accumulation of intracellular fatty acids also negatively impacts the activity of SaeRS. Incubation of S. aureus with P. aeruginosa cell-free conditioned culture medium decreased the transcriptional output of the SaeRS system. Further analyses using P. aeruginosa mutant strains and chemical genetics determined that 2-heptyl-4-quinolone N-oxide (HQNO) was responsible for the SaeRS-dependent changes in gene regulation. Treatment with HQNO increased the abundance of cell-associated fatty acids. HQNO inhibits cell respiration, and the SaeRS system did not respond to HQNO treatment in a respiration-impaired S. aureus strain, which accumulates fatty acids. The data presented are consistent with a working model wherein treatment of S. aureus with HQNO inhibits respiration, increasing free fatty acid accumulation, which negatively impacts SaeRS signaling. This results in decreased expression of the SaeRS regulon, which has significant roles in pathogenesis.IMPORTANCEPseudomonas aeruginosa and Staphylococcus aureus are often co-isolated from the airways of cystic fibrosis patients. P. aeruginosa secretes non-essential metabolites that alter S. aureus physiology, providing P. aeruginosa with a competitive advantage. S. aureus can adapt to the presence of these metabolites, but the genetic mechanisms used to sense these P. aeruginosa-produced metabolites and/or the induced physiological changes are largely unknown. The S. aureus SaeRS two-component regulatory system positively regulates the expression of various virulence factors, including toxins and proteases, that facilitate adaptation to and survival in hostile host environments. This study demonstrates that the P. aeruginosa-produced respiratory toxin 2-heptyl-4-quinolone N-oxide inhibits respiration, decreasing the transcription of SaeRS-regulated genes and thereby decreasing virulence factor production. These findings could be exploited to decrease the ability of S. aureus to express virulence factors in various infection settings.

Individuals with poorly controlled diabetes mellitus often develop multispecies skin and soft tissue infections, with Staphylococcus aureus and Pseudomonas aeruginosa among the most prevalent bacteria isolated from infection sites worldwide. Diabetic infections are recalcitrant to conventional antibiotic regimens and may be a reservoir for emergent antibiotic-resistant bacterial strains. Supporting this, we have previously shown that rifampicin treatment elicits the emergence and expansion of rifampicin-resistant (Rif-r) S. aureus only in diabetic mice, potentially due to greater bacterial outgrowth increasing the frequency of resistance-conferring mutations. However, whether S. aureus exhibits altered resistance outcomes during multispecies diabetic infections is unclear. During co-infection with P. aeruginosa under normoglycemic conditions, S. aureus exhibits reduced growth and altered susceptibility to several antibiotics. In contrast, we previously observed that glucose availability allows S. aureus to largely overcome P. aeruginosa-mediated growth inhibition. Here, we explored S. aureus resistance outcomes under hyperglycemic conditions in the context of co-infection with P. aeruginosa during antibiotic challenge. We found that P. aeruginosa exoproducts regulated by the Pseudomonas quinolone signal quorum sensing system inhibit the emergence but not the expansion of Rif-r S. aureus in vitro under glucose-replete conditions. In contrast, we recovered equivalent Rif-r S. aureus burdens from diabetic mice during mono- and co-infection with P. aeruginosa. These results demonstrate that the diabetic infection microenvironment is conducive to emergent Rif-r S. aureus despite external pressures elicited by P. aeruginosa.IMPORTANCEPoorly controlled diabetes mellitus confers an increased susceptibility to bacterial infections, with Staphylococcus aureus and Pseudomonas aeruginosa frequently isolated from diabetic skin wounds. S. aureus readily develops antibiotic resistance during diabetic mono-infection under antibiotic pressure, but whether this occurs during diabetic co-infection is unclear. Under normoglycemic conditions, secreted P. aeruginosa factors alter S. aureus tolerance to several antibiotics. Here, we show that these P. aeruginosa exoproducts further inhibit the emergence of antibiotic-resistant S. aureus regardless of glucose availability in vitro, but this does not occur during subcutaneous co-infection in diabetic mice. These results provide initial insights regarding conditions that may inhibit S. aureus resistance development in hyperglycemic environments but underscore the influence of the host infection microenvironment in shaping resistance outcomes.

The aminoacyltransferase MurM is an important penicillin resistance determinant in Streptococcus pneumoniae. This enzyme attaches a serine or alanine to the side chain of lysine, the third residue of the pentapeptide of lipid II, resulting in branched muropeptides that can be crosslinked to stem peptides in peptidoglycan by penicillin binding proteins (PBPs). Deletion of murM results in only linear muropeptides, and more importantly, a significant reduction in resistance. Highly penicillin-resistant pneumococci express low-affinity PBPs, an altered MurM protein, and possess a highly branched cell wall. It has therefore been hypothesized that MurM, and thus branched muropeptides, are essential for resistance because they are better substrates for low-affinity PBPs. In this study, we found that neither the version of murM nor elevated levels of cell wall branching affected resistance levels. To further support this, we investigated whether branched muropeptide substrates compete better than linear versions with penicillin at the active site of low-affinity PBPs and quantified changes to the stem peptide composition of the resistant Pen6 strain in response to subinhibitory concentrations of penicillin. We found that the level of cell wall branching decreased during penicillin exposure. Together, our results do not support the idea that elevated levels of branched muropeptides (more active MurM) are important for either the function of low-affinity PBPs or the cell's response to penicillin. Nevertheless, since a functional MurM enzyme is important for resistance, we speculate that it might indirectly influence other functions related to cell wall synthesis and remodeling needed for a resistant phenotype.IMPORTANCEA fundamental understanding of the mechanisms behind antibiotic resistance is needed to find strategies to extend the clinical relevance of existing drugs. This study explores the relationship between cell wall composition and penicillin resistance in Streptococcus pneumoniae. Here, we confirm that branched peptide crosslinks in the cell wall are crucial for resistance but found no correlation between elevated branching levels and resistance. Our data suggest that the function of low-affinity penicillin binding proteins is not influenced by the lack of branched cell wall precursors. Instead, a branched cell wall might contribute to resistance via other cell wall biosynthesis and remodeling mechanisms. These insights could offer new perspectives on why a branched cell wall is important for penicillin resistance in pneumococci.

The opportunistic human pathogen Pseudomonas aeruginosa (Pa), a leading cause of severe infections, becomes increasingly resistant to antibiotics, including the last resort antibiotic, polymyxin B (PMB). Previous studies have shown that calcium (Ca²⁺) at the levels encountered during infections increases Pa resistance to PMB. However, the mechanisms of this Ca²⁺ regulation are not known. Here, we identified three novel genes (PA2803, PA3237, and PA5317) that contribute to the Ca²⁺-dependent PMB resistance in Pa. PA2803, the focus of this work, encodes a putative phosphonatase and is a founding member of the PA2803 subfamily from the Haloacid Dehalogenase Superfamily. Since the transcription of this gene is regulated by both Ca²⁺ and inorganic phosphate (P_i), we named it "P_i and Ca²⁺-regulated protein, PcrP." Congruent with sequence-based predictions, we showed that PcrP lacks catalytic activity and instead binds protein partners, revealing a novel non-catalytic function. By using pull-down assays and bacterial two-hybrid systems, we identified and validated two protein partners of PcrP: Acp3 and PA3518. We showed that PcrP is involved in oxidative stress responses in Pa, which are likely mediated by its interactions with Acp3 and may support its role in PMB resistance. In addition, PcrP imparts a Ca²⁺-dependent growth advantage during P_i starvation and plays a role in polyphosphate accumulation in a Ca²⁺-dependent manner. Overall, this study identified a novel protein-binding function for the PA2803 subfamily representative, which mediates Pa responses to elevated Ca²⁺ and P_i starvation and enhances PMB resistance.IMPORTANCEPseudomonas aeruginosa (Pa) is a critical human pathogen that presents significant clinical challenges, underscoring the urgent need for understanding its resistance mechanisms. Previous studies have shown that calcium (Ca²⁺) at the levels detected during infections increases Pa resistance to the last resort antibiotic polymyxin B (PMB). For the first time, we identified three novel genes, whose products are required for the Ca²⁺-dependent PMB resistance in Pa. One of them, PA2803, regulated by Ca²⁺ and phosphate, was named phosphate and Ca²⁺-regulated protein, PcrP. This study discovered a novel protein-binding function of PcrP and identified two protein partners. Given the high level of sequence conservation within the PA2803 subfamily, the protein-binding function may be shared by other members of the PA2803 subfamily.

The L-glucose catabolic pathway of Luteolibacter sp. strain LG18 was determined. L-glucose dehydrogenase (LguA) and L-gluconate dehydrogenase (LguD), purified from the cell extract of strain LG18, convert L-glucose to 5-keto-L-gluconate via L-gluconate, and these recombinant enzymes also utilize L-galactose and L-galactonate, respectively. Genes encoding these enzymes are both located in the gene cluster, lguABCDEF, which includes other genes possibly involved in L-galactose catabolism. After oxidation of L-gluconate, 5-keto-L-gluconate is converted to D-tagaturonate by LguG, a C-4 epimerase, determined with the recombinant enzyme. The subsequent LG18 reactions are likely to proceed in the same way as Escherichia coli L-galactonate catabolism, wherein LguC reduces C-5 to produce D-altronate that is dehydrated by LguB to produce 2-keto-3-deoxy-D-gluconate (KDG). LguH then phosphorylates KDG C-6 to produce KDG-6-phosphate, and an aldolase reaction driven by LguE produces D-glyceraldehyde-3-phosphate and pyruvate. Both lguG and lguH lie outside the lguABCDEF cluster, and LguH had a novel preference in utilizing pyrophosphate as a phosphate donor rather than ATP. Gene disruption studies indicated that, with the exception of lguG, which is involved only in L-glucose catabolism, the identified genes are indeed responsible for both L-glucose and L-galactose catabolism, indicative of a dual L-glucose/L-galactose catabolic pathway governed by a single set of genes. All the orthologs in this pathway are conserved in several Luteolibacter species, which also utilize L-glucose, suggesting that the same catabolic pathway is present in this genus.IMPORTANCEL-glucose is presumably not present in natural environments, and to date, L-glucose catabolism has only been reported for a Paracoccus laeviglucosivorans strain 43P. The Luteolibacter strain LG18 differs taxonomically from 43P at the phylum level, and its L-glucose catabolic pathway differs from that of 43P at later steps from the C-4 epimerization reaction. In addition, most genes that drive LG18 L-glucose catabolism are also responsible for L-galactose catabolism, indicating the presence of a dual L-glucose/L-galactose catabolic pathway. This report contributes to a better understanding of homochirality in sugar catabolism, especially catabolism of glucose.

Pseudomonas aeruginosa is a highly adaptable bacterial pathogen with a resilient cell envelope. This envelope must be elongated as cells grow, which requires coordinated biosynthesis of the inner and outer membranes and the peptidoglycan cell wall. Cell wall endopeptidases are essential to expand the peptidoglycan sacculus, and the LbcA•CtpA proteolytic complex controls the activity of multiple endopeptidases by degrading them. Here, we report an investigation into control of the LbcA•CtpA proteolytic complex and its substrates. LbcA and CtpA levels were unaffected by growth rate, which corresponded with constitutive expression of their genes. For CtpA, this was explained by its arrangement in a complex operon containing an internal ctpA promoter. Despite constitutive LbcA and CtpA production, the LbcA•CtpA substrate levels were higher when cells were growing rapidly. In most cases, this correlated with modestly higher substrate gene expression in the exponential phase. However, most of the control came from reduced CtpA activity when cells were growing rapidly. Our data suggest that CtpA activity might be affected by phospholipid transport and related processes in the cell envelope. A similar phenomenon was reported to affect the Escherichia coli NlpI•Prc complex, even though there are major sequence and structural differences between the NlpI•Prc and LbcA•CtpA complexes. This makes it likely that growth-rate-dependent autolysin control by these proteolytic complexes is widely conserved, even if they are composed of non-orthologous proteins in some cases.IMPORTANCECarboxyl-terminal processing proteases occur in all domains of life. Some are associated with bacterial virulence, including P. aeruginosa CtpA, which works with the outer membrane lipoprotein LbcA to degrade cell wall endopeptidases. We report that the LbcA•CtpA complex activity is coordinated with growth rate, ensuring appropriate levels of its substrates for cell wall expansion. The mechanism appears to be connected to phospholipid transport, much like a phenomenon reported for Escherichia coli NlpI•Prc complex. However, the NlpI•Prc and LbcA•CtpA complexes are not orthologs. Therefore, growth-rate-dependent control by analogous but dissimilar complexes might be a widely conserved mechanism, and one that could perhaps be targeted for therapeutic intervention.

Bacillus subtilis is able to catabolize fructosamines, also known as Amadori rearrangement products. The frlBONMD-frlP operon mediates this process and is subjected to specific and global regulation. Although the degradation pathway favoring α-glycated amino acids is known, the mechanisms of substrate uptake have remained unclear. In this study, mutagenic and functional analyses revealed that FrlONM, a type I ABC importer, along with the nucleotide-binding domain (NBD) FrlP, is required for the uptake of fructosevaline. Transcriptional and translation frlP-lacZ fusions indicated that frlP is induced by fructosevaline and negatively regulated by the FrlR repressor. In addition, we show that MsmX, a multitask NBD of B. subtilis, is also able to serve as an energy motor of this type I ABC importer and that its presence alongside FrlP is vital for optimal growth on fructosevaline. To address the physiological significance of this functional redundancy, we assessed the distribution of ABC type I NBDs FrlP and MsmX across the Bacillaceae family. MsmX is homogeneously distributed in the Bacillaceae family tree, while FrlP is restricted to the Bacillus subtilis group, suggesting that the presence of FrlP together with other components of the fructosamines operon is important for bacterial fitness in plant-associated ecological niches.IMPORTANCEBacillus subtilis is widely applied in the industry as a microbial cell factory, as a biofertilizer for sustainable agriculture, in the animal feed industry and as human probiotic. In its natural environment, B. subtilis helps to shape the gut microbiome and the phytomicrobiome. Fructosamines, or Amadori rearrangement products, are ubiquitously found in nature and serve as precursors of toxic cell end-products implicated in the pathology of human diseases. This study provides a solid contribution to a deep knowledge of transport mechanisms, genetic regulation, and physiological relevance of fructosamines utilization in B. subtilis. Moreover, it highlights an unusual strategy to adapt to alterations in nutrient availability by swapping the energy providing domain of ABC transporters.

Two-component regulatory systems typically consist of a sensor kinase and a response regulator. Phosphorylation of the receiver domain controls response regulator activity. Pseudo-receivers (PsRs) are identified computationally as receivers but lack key residues to catalyze phosphotransfer reactions. Although PsRs are common, molecular mechanisms that activate and inactivate bacterial PsRs remain a mystery. We untangled four potentially related but distinct concepts: bacterial PsRs, receivers with regulatory mechanisms in addition to phosphorylation, receivers that are active without phosphorylation, and orphan receivers without an obvious partner sensor kinase. We also analyzed bacterial PsR sequences and structures to identify regions of likely functional significance.

Thiamine pyrophosphate (TPP)-responsive riboswitches are genetic elements in bacteria that regulate the expression of genes coding for proteins involved in the biosynthesis and transport of thiamine (vitamin B₁). Following uptake, cytoplasmic thiamine is converted to TPP, which serves as a cofactor for enzymes of central metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway, and it is the level of TPP (and not thiamine) that is sensed by TPP riboswitches. TPP riboswitches are the most widespread riboswitches in bacteria. Their key roles in metabolism combined with their absence in humans make them potential targets for antibiotics, whereby the focus of the present study was pathogenic bacteria of the ESKAPE group: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. As a first step toward the development of novel TPP riboswitch-targeting antimicrobials to treat infections caused by ESKAPE organisms, we characterized various TPP riboswitches present in these bacteria. We developed a dual-luciferase reporter gene assay to monitor riboswitch activity and found that most of the predicted TPP riboswitches indeed were functional regulators and responded to TPP. In contrast to the Escherichia coli thiC TPP riboswitch, TPP riboswitches from ESKAPE bacteria were found not to respond to the synthetic thiamine analog pyrithiamine. One TPP riboswitch of K. pneumoniae was examined in detail with regard to the effect of pyrithiamine. Site-directed mutagenesis experiments identified specific nucleotides responsible for the non-response to pyrithiamine, and this should be useful in developing novel TPP riboswitch-targeting antimicrobials.

Importance: Riboswitches are RNA molecules that control important processes in bacteria. Infections with pathogens of the ESKAPE group are common, and we are trying to find new ways to fight these bacteria. Small molecules can be designed to bind to riboswitches and optimally block their activity. In the present work, we have analyzed the thiamine pyrophosphate (TPP) riboswitches of ESKAPE pathogens with respect to small molecule binding. For this purpose, we developed a dual-luciferase reporter gene assay. Most of the predicted TPP riboswitches were indeed functional regulators and are thus targets for new anti-infectives. The small molecule pyrithiamine does not block all TPP riboswitches tested, and we found a structural basis for this behavior.

Bacteroidales is an order of bacteria that includes members that colonize the human gut, oral cavity, cow rumen, and other host-associated environments. Most humans become colonized with gut Bacteroidales species relatively soon after birth and later become colonized at high density with numerous diverse species. Bacteroidales strains often persist in the human gut for decades where they extensively evolve, acquiring point mutations, prophage, mobile plasmids, and integrative conjugal elements, making each person's gut Bacteroidales strains highly personalized. Much of what we have learned about basic biological properties of gut Bacteroidales comes from analyses of two species, Bacteroides fragilis and Bacteroides thetaiotaomicron, which were studied for different reasons. Three decades ago, there was only one human gut Bacteroidales genus recognized, the Bacteroides, into which all human gut Bacteroidales species were classified. Today, the human gut Bacteroidales number over 50 species with more than 14 genera and at least seven families. Studies of B. fragilis and B. thetaiotaomicron have provided a wealth of information of basic processes of these gut symbionts, many of which are generally applicable to other species of gut Bacteroidales. In this review, I provide a historical perspective as to why these two species have served as models, as well as some of the biological processes learned from studies of these two species. Finally, I discuss why present and future analyses of the gut Bacteroidales have expanded beyond these two model organisms.