The importance of MpsABC for bacterial growth under atmospheric CO2 levels

Almost 50 years ago, carbon dioxide (CO2) dependent variants were described in non-autotrophic bacteria. However, the underlying mechanism of CO2-dependency are not clear. In this study, we demonstrate that the Staphylococcus aureus mpsAB operon does not only contribute to the production of membrane potential but is also vital for growth under atmospheric CO2 concentration. Deletion mutants of mpsA, mpsB, and mpsABC hardly grew under atmospheric air conditions but could be restored to near wild-type levels under elevated CO2 or bicarbonate concentrations. Uptake studies with radiolabeled sodium bicarbonate (NaH14CO3) revealed that MpsAB represents a bicarbonate/dissolved inorganic carbon (DIC) transporter. Additionally, an Escherichia coli carbonic anhydrase (CA) can mutant which is unable to grow under atmospheric air but can grow when elevated CO2 is present, can be complemented by S. aureus mpsAB and vice versa. This mutual complementation experiments indicate that both the DIC transporter and CA represent a DIC concentrating system that can functionally substitute each other. Compared to the wild type, production of hemolytic toxins are less in S. aureus mps mutants and they also display less virulence in mouse model of infection. Phylogenetic analysis reveals that MpsAB homologs are ubiquitous in bacteria and frequently occur side-by-side on the genome. Interestingly, the majority of bacteria possess homologs of either MpsAB or CA, while both are present in some highly pathogenic species. Taken together, MpsAB is proposed to act as a DIC transporter or bicarbonate concentrating system, potentially operating as a sodium bicarbonate cotransporter. In a separate work, the mechanisms behind the host cell internalization triggered by Lpl proteins was elucidated. Lipoprotein-like lipoproteins (Lpls) is encoded by the S. aureus lpl cluster on a pathogenicity island named νSaα island which consists of 10 lpls paralogue genes. By using recombinant Lpl1 protein as a model of Lpls, the host receptor for Lpl-induced S. aureus USA300 invasion of human keratinocytes was identified as human heat shock protein Hsp90 via pull-down assay. Synthetic peptides which covers the Lpl1 sequence resulted in double to five-fold higher rate of S. aureus invasion in HaCaT cells and in primary human keratinocytes. Lpl-Hsp90 interaction stimulates F-actin formation, leading to an endocytosis-like engulfment of S. aureus

The MpsAB Bicarbonate Transporter Is Superior to Carbonic Anhydrase in Biofilm-Forming Bacteria with Limited CO 2 Diffusion

CO2 and bicarbonate are required for carboxylation reactions, which are essential in most bacteria. To provide the cells with sufficient CO2, there exist two dissolved inorganic carbon supply (DICS) systems: the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA). Recently, it has been shown that MpsAB is a bicarbonate transporter that is present not only in photo- and auto-trophic bacteria, but also in a diverse range of nonautotrophic microorganisms. Since the two systems rarely coexist in a species but are interchangeable, we investigated what advantages the one system might have over the other. Using the genus Staphylococcus as a model, we deleted the CA gene can in Staphylococcus car-nosus and mpsABC genes in Staphylococcus aureus. Deletion of the respective gene in one or the other species led to growth inhibition that could only be reversed by CO2 supplementation. While the S. carnosus Δcan mutant could be fully complemented with mpsABC,the S. aureus ΔmpsABC mutant was only partially complemented by can, suggesting that MpsAB outperforms CA. Interestingly, we provide evidence that mucus biofilm formation such as that involving polysaccharide intercellular adhesin (PIA) impedes the diffusion of CO2 into cells, making MpsAB more advantageous in biofilm-producing strains or species. Coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. In conclusion, the distribution of MpsAB or CA in bacteria does not appear to be random as expression of bicarbonate transporters provides an advantage where diffusion of CO2 is impeded. IMPORTANCE CO2 and bicarbonate are required for carboxylation reactions in central metabolismandbiosynthesisofsmallmoleculesinallbacteria.Thisisachievedbytwo different systems for dissolved inorganic carbon supply (DICS): these are the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA), but both rarely coexist in a given species. Here, we compared both systems and demonstrate that the distribution of MpsAB and/or CA within the phylum Firmicutes is apparently not random. The bicarbonate transporter MpsAB has an advantage in species where CO2 diffusion is hampered—for instance, in mucus- and biofilm-forming bacteria. However, coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. Given the clinical relevance of Staphylococcus in the medical environment, such findings contribute to the understanding of bacterial metabolism and thus are crucial for exploration of potential targets for antimicrobials. The knowledge gained here as exemplified by staphylococcal species could be extended to other pathogenic bacteria.Fil: Fan, Sook Ha. Eberhard Karls Universität Tübingen.; AlemaniaFil: Matsuo, Miki. Eberhard Karls Universität Tübingen.; AlemaniaFil: Huang, Lili. Eberhard Karls Universität Tübingen.; AlemaniaFil: Tribelli, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Götz, Friedrich. Eberhard Karls Universität Tübingen.; Alemani

the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine

Funding Information: This work was supported by the following grant from the National Institutes of Health: 1RO1-AI146078 (to A.S.B.). Conceptualization: S.C.E., A.S.B., R.A.P.; methodology: S.C.E., W.E.R., W.A.; formal analysis: S.C.E., W.E.R., W.A.; investigation: S.C.E., W.E.R., W.A., S.H.F., S.L.M., A.M.E.; resources: A.S.B.; writing – original draft: S.C.E., A.S.B.; writing – review and editing: S.C.E., A.S.B., R.A.P., H.F.C.; supervision: A.S.B.; project administration: A.S.B.; funding acquisition: A.S.B. Funder Grant(s) Author(s) HHS | National Institutes of Health (NIH) AI146078 Arnold S. Bayer Funding Information: This work was supported by the following grant from the National Institutes of Health: 1RO1-AI146078 (to A.S.B.). Publisher Copyright: Copyright © 2024 Ersoy et al.Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most β-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed “NaHCO3-responsiveness” has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care β-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such “NaHCO3-responsive” isolates can be effectively cleared by β-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting β-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific β-lactam agents.publishersversionpublishe

The presence of high level soluble herpes virus entry mediator in sera of gastric cancer patients

The development of gastric cancer (GC) is closely related to chronic inflammation caused by Helicobacter pylori infection, and herpes virus entry mediator (HVEM) is a receptor expressed on the surface of leukocytes that mediates potent inflammatory responses in animal models. However, the role of HVEM in human GC has not been studied. Previously, we showed that the interaction of HVEM on human leukocytes with its ligand LIGHT induces intracellular calcium mobilization, which results in inflammatory responses including induction of proinflammatory cytokine production and anti-bacterial activities. In this study, we report that leukocytes from GC patients express lower levels of membrane HVEM (mHVEM) and have lower LIGHT-induced bactericidal activities than those from healthy controls (HC). In contrast, levels of soluble HVEM (sHVEM) in the sera of GC patients were significantly higher than in those of HC. We found that monocyte membrane-bound HVEM is released into the medium when cells are activated by proinflammatory cytokines such as TNF-α and IL-8, which are elevated in the sera of GC patients. mHVEM level dropped in parallel with the release of sHVEM, and release was completely blocked by the metalloprotease inhibitor, GM6001. We also found that the low level of mHVEM on GC patient leukocytes was correlated with low LIGHT-induced bactericidal activities against H. pylori and S. aureus and production of reactive oxygen species. Our results indicate that mHVEM on leukocytes and sHVEM in sera may contribute to the development and/or progression of GC

Surface Properties of Helicobacter pylori Urease Complex Are Essential for Persistence

The enzymatic activity of Helicobacter pylori's urease neutralises stomach acidity, thereby promoting infection by this pathogen. Urease protein has also been found to interact with host cells in vitro, although this property's possible functional importance has not been studied in vivo. To test for a role of the urease surface in the host/pathogen interaction, surface exposed loops that display high thermal mobility were targeted for inframe insertion mutagenesis. H. pylori expressing urease with insertions at four of eight sites tested retained urease activity, which in three cases was at least as stable as was wild-type urease at pH 3. Bacteria expressing one of these four mutant ureases, however, failed to colonise mice for even two weeks, and a second had reduced bacterial titres after longer term (3 to 6 months) colonisation. These results indicate that a discrete surface of the urease complex is important for H. pylori persistence during gastric colonisation. We propose that this surface interacts directly with host components important for the host-pathogen interaction, immune modulation or other actions that underlie H. pylori persistence in its special gastric mucosal niche

Association of fat mass and obesity-associated (FTO) gene rs9939609 variant with obesity among multi-ethnic Malaysians in Kampar, Perak

Obesity is a multifactorial disease caused by the interaction of genetic, lifestyle and environmental factors. Common single nucleotide polymorphisms in the recently-described Fat Mass and Obesity-Associated (FTO) gene have been related to body weight and fat mass in humans and genome-wide association studies in several populations have indicated that the FTO rs9939609 variant is associated with obesity. Therefore, the objective of this study was to investigate the association of the FTO rs9939609 variant with obesity among 324 multi-ethnic Malaysians (98 Malays, 158 Chinese, 68 Indians) who attended the Kampar Health Clinic, Perak. With the overall minor A allele frequency (MAF) of 0.199, the distribution of genotypes and alleles was significantly different among ethnicities (MAF highest among Malays), but no association was found for obesity, related anthropometric measurements and gender. Subject with allele A had marginally but significantly higher waist circumference (p=0.015), thus the FTO rs9939609 allele was associated with central obesity (p=0.034 by Chi-square analysis; Odds Ratio (OR)=1.680 (CI=1.036, 2.724; p=0.035)). However, this association was abolished when adjusted for age, gender and ethnicity (OR=1.455, CI=0.874, 2.42; p=0.149). In conclusion, the MAF of the FTO rs9939609 SNP was low as in other Asian populations and there was no evidence for an involvement of this SNP in obesity and obesity-related traits in this multi-ethnic Malaysian study group

Staphylococcus aureus Genomes Harbor Only MpsAB-Like Bicarbonate Transporter but Not Carbonic Anhydrase as Dissolved Inorganic Carbon Supply System

In recent years, it became apparent that not only autotrophic but also most other bacteria require CO(2) or bicarbonate for growth. Two systems are available for the acquisition of dissolved inorganic carbon supply (DICS): the cytoplasmic localized carbonic anhydrase (CA) and the more recently described bicarbonate transporter MpsAB (membrane potential generating system). In the pathogenic species Staphylococcus aureus, there are contradictions in the literature regarding the presence of a CA or MpsAB. Here, we address these contradictions in detail. We could demonstrate by careful BLASTp analyses with 259 finished and 4,590 unfinished S. aureus genomes that S. aureus does not contain CA and that the bicarbonate transporter MpsAB is the only DICS system in this species. This finding is further supported by two further pieces of evidence: (i) mpsAB deletion mutants in four different S. aureus strains failed to grow under atmospheric air, which should not be the case if they possess CAs, since we have previously shown that both CA and MpsAB can substitute for each other, and (ii) S. aureus is completely resistant to CA inhibitors, whereas Staphylococcus carnosus, which has been shown to have only CA, was inhibited by ethoxyzolamide (EZA). Taken together, we demonstrate beyond doubt that the species S. aureus possesses only the bicarbonate transporter MpsAB as its sole DICS system. IMPORTANCE The discrepancies in the current literature and even in NCBI database, which listed some protein sequences annotated as Staphylococcus aureus carbonic anhydrase (CA), are misleading. One of the existing problems in publicly available sequence databases is the presence of incorrectly annotated genes, especially if they originated from unfinished genomes. Here, we demonstrate that some of these unfinished genomes are of poor quality and should be interpreted with caution. In the present study, we aimed to address these discrepancies and correct the current literature about S. aureus CA, considering the medical relevance of S. aureus. If left unchecked, these misleading studies and wrongly annotated genes might lead to a continual propagation of wrong annotation and, consequently, wrong interpretations and wasted time. In addition, we also show that bicarbonate transporter MpsAB-harboring bacteria are resistant to CA inhibitor, suggesting that pathogens possessing both MpsAB and CA are not treatable with CA inhibitors

Association of the Cocaine- and Amphetamine-Regulated Transcript Prepropeptide Gene (CARTPT) rs2239670 Variant with Obesity among Kampar Health Clinic Patrons, Malaysia

Background: Cocaine- and amphetamine-regulated transcript (CART) is a hypothalamic anorectic neuropeptide that controls feeding behaviour and body weight. The study objective was to investigate the association of the CART prepropeptide gene (CARTPT) rs2239670 variant with obesity and its related anthropometric indicators among patients of a Malaysian health clinic in Kampar, Perak, Malaysia. Methods: A total of 300 Malay/Peninsular Bumiputera, Chinese, and Indian subjects (115 males, 185 females; 163 non-obese, 137 obese) were recruited by convenience sampling, and anthropometric measurements, blood pressures, and pulse rate were taken. Genotyping was performed using AvaII polymerase chain reaction–restriction fragment length polymorphism. Results: Genotyping revealed 203 (67.7%), 90 (30.0%), and 7 (2.3%) subjects with the GG, GA, and AA genotypes, respectively, with a minor allele (A) frequency of 0.17. No significant difference in the CARTPT rs2239670 genotype and allele distribution was found between obese and non-obese subjects, and logistic regression showed no association between the mutated genotypes (GA, AA) and allele (A) with obesity, even after adjusting for age, gender, and ethnicity. Furthermore, the measurements did not differ significantly between the genotypes and alleles. No significant difference in the genotype and allele distribution was found among genders, but they were significantly different among ethnicities (P = 0.030 and P = 0.019, respectively). Conclusion: CARTPT rs2239670 is not a predictor for obesity among the Malaysian subjects in this study

The MpsAB Bicarbonate Transporter Is Superior to Carbonic Anhydrase in Biofilm-Forming Bacteria with Limited CO2 Diffusion

CO(2) and bicarbonate are required for carboxylation reactions, which are essential in most bacteria. To provide the cells with sufficient CO(2), there exist two dissolved inorganic carbon supply (DICS) systems: the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA). Recently, it has been shown that MpsAB is a bicarbonate transporter that is present not only in photo- and autotrophic bacteria, but also in a diverse range of nonautotrophic microorganisms. Since the two systems rarely coexist in a species but are interchangeable, we investigated what advantages the one system might have over the other. Using the genus Staphylococcus as a model, we deleted the CA gene can in Staphylococcus carnosus and mpsABC genes in Staphylococcus aureus. Deletion of the respective gene in one or the other species led to growth inhibition that could only be reversed by CO(2) supplementation. While the S. carnosus Δcan mutant could be fully complemented with mpsABC, the S. aureus ΔmpsABC mutant was only partially complemented by can, suggesting that MpsAB outperforms CA. Interestingly, we provide evidence that mucus biofilm formation such as that involving polysaccharide intercellular adhesin (PIA) impedes the diffusion of CO(2) into cells, making MpsAB more advantageous in biofilm-producing strains or species. Coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. In conclusion, the distribution of MpsAB or CA in bacteria does not appear to be random as expression of bicarbonate transporters provides an advantage where diffusion of CO(2) is impeded. IMPORTANCE CO(2) and bicarbonate are required for carboxylation reactions in central metabolism and biosynthesis of small molecules in all bacteria. This is achieved by two different systems for dissolved inorganic carbon supply (DICS): these are the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA), but both rarely coexist in a given species. Here, we compared both systems and demonstrate that the distribution of MpsAB and/or CA within the phylum Firmicutes is apparently not random. The bicarbonate transporter MpsAB has an advantage in species where CO(2) diffusion is hampered—for instance, in mucus- and biofilm-forming bacteria. However, coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. Given the clinical relevance of Staphylococcus in the medical environment, such findings contribute to the understanding of bacterial metabolism and thus are crucial for exploration of potential targets for antimicrobials. The knowledge gained here as exemplified by staphylococcal species could be extended to other pathogenic bacteria