Biology subjects

Hines, K. M.

Publications and source records attributed to Hines, K. M..

13 recordsLinked to original sources

bioRxiv2026

Exploring the Impact of S. aureus Lipase Activity on Intra- and Extracellular Lipids

Previous research has shown that mammalian fatty acids (FAs) can influence antibiotic tolerance of Staphylococcus aureus, yet many of these studies overlook the sources of these FAs, which are primarily esterified into glycero- and phospholipids, and the impact S. aureus lipase activity has on host lipids. Here we attempt to gain insight into the complex interplay between the S. aureus lipidome and its environment using culture media supplemented tissue-specific phospholipid mixtures. Phospholipid profiles of heart, liver, and brain-derived lipids revealed distinct distributions of headgroup and fatty acyl tail structures within the phospholipids. Following the growth of S. aureus in lipid-enriched broth, PG species containing mono- and poly-unsaturated acyl tails were detected with abundances that correlated strongly with the FA profile of the tissue extract. We found that S. aureus cultured with liver-derived lipid extract, which yielded the most unsaturated PGs, promoted growth in high concentrations of the membrane-targeting antimicrobial daptomycin. To explore the influence of lipase activity on the extracellular lipids, comparative analysis of fresh versus spent media revealed that the lipase-mediated degradation of complex phospholipid mixtures was influenced by both head group structure and acyl tail linkage. Concurrently, the spent media contained elevated levels of mono- and polyunsaturated lysophospholipids that were predominantly of the 2-acyl form rather than the 1-acyl form observed in the fresh media. Together, these results demonstrate the extent to which the lipase activity of S. aureus remodels both its own lipidome as well as the structures of the phospholipids in the surrounding environment. IMPORTANCES. aureus releases a secreted glycerol ester hydrolase, Geh, into the extracellular environment, which enables the bacterium to generate free FA from glycerolipids, phospholipids, and cholesterol esters that are present in surrounding tissue of an infection. The liberated FAs can be incorporated into the phospholipids of S. aureus, thereby altering its membrane physiology with mono- and poly-unsaturated FAs it cannot otherwise synthesize. Simultaneously, the action of Geh on lipids in the host environment leads to higher levels of bioactive lysophospholipids that participate in mammalian signaling pathways. This work reveals the preferences of S. aureus Geh across phospholipids with different head group and acyl tail structures found within tissue-derived lipid extracts, as well as the fate of the liberated FAs within the staphylococcal membrane lipids. The impacts of these processes on both the host and bacterium have implications for the immune response to and antibiotic treatment of S. aureus infections.

microbiology↗

bioRxiv2026

Enhancement of low-temperature growth of Staphylococcus aureus by low concentrations of antimicrobial unsaturated fatty acids

The incorporation of oleic acid, a physiologically relevant and relatively non-toxic fatty acid, into membrane phospholipids and glycolipids has major impacts on the membrane biophysical properties of Staphylococcus aureus, including the promotion of low-temperature growth. Inclusion of oleic acid in growth media decreased the minimum growth temperature of S. aureus. It has not been studied yet whether other physiologically relevant, yet more antimicrobial straight-chain unsaturated fatty acids (SCUFAs), elicit similar responses. Here, we report that exogenous addition of these antimicrobial SCUFAs at 12{degrees}C led to their incorporation into the membrane lipids in only relatively small amounts, which, however, was sufficient to strongly promote growth at low temperatures. Intriguingly, these SCUFAs showed a preferential incorporation into diglucosyldiacylglyceride (DGDG) over phosphatidyl glycerol, and increased DGDG levels [~] 4-fold in SCUFA-grown S. aureus. Two glycolipid-deficient mutants lost the growth-enhancing effects of SCUFAs at low temperatures and showed increased susceptibility to SCUFAs at 37{degrees}C, suggesting that glycolipids are important membrane components and are required for low-temperature adaptation. The presence of SCUFAs at low temperatures also enhanced the production of the carotenoid staphyloxanthin. Overall, our results suggest that multiple strategies are at play in the membrane lipid remodeling of S. aureus to adapt to the stress of low-temperature growth and likely to other environmental stresses. Incorporation of antimicrobial SCUFAs into membrane lipids may facilitate the insertion of free forms of these SCUFAs into the membrane and be a part of their mode of action.

microbiology↗

bioRxiv2024

Commensal-derived short-chain fatty acids disrupt lipid membrane homeostasis in Staphylococcus aureus

The role of commensal anaerobic bacteria in chronic respiratory infections is unclear, yet they can exist in abundances comparable to canonical pathogens in vivo. Their contributions to the metabolic landscape of the host environment may influence pathogen behavior by competing for nutrients and creating inhospitable conditions via toxic metabolites. Here, we reveal a mechanism by which the anaerobe-derived short chain fatty acids (SCFAs) propionate and butyrate negatively affect Staphylococcus aureus physiology by disrupting branched chain fatty acid (BCFA) metabolism. In turn, BCFA impairment results in impaired growth, diminished expression of the agr quorum sensing system, as well as increased sensitivity to membrane-targeting antimicrobials. Altered BCFA metabolism also reduces S. aureus fitness in competition with Pseudomonas aeruginosa, suggesting that airway microbiome composition and the metabolites they produce and exchange directly impact pathogen succession over time. The pleiotropic effects of these SCFAs on S. aureus fitness and their ubiquity as metabolites in animals also suggests that they may be effective as sensitizers to traditional antimicrobial agents when used in combination.

microbiology↗

bioRxiv2024

Microglia Morphological Response to Mesenchymal Stromal Cell Extracellular Vesicles Demonstrates EV Therapeutic Potential for Modulating Neuroinflammation

BackgroundMesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation-relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. ResultsUsing an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-{gamma}) and tumor necrosis factor-alpha (TNF-). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-{gamma}/IFN- stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. ConclusionThis study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphological approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

bioengineering↗

bioRxiv2024

Growth of Staphylococcus aureus in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides

Staphylococcus aureus readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, S. aureus presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When S. aureus is grown conventionally, its membrane lipids contain a mix of branched-chain and straight-chain saturated fatty acids. However, when unsaturated fatty acids are present in the growth medium, they become a major part of the total fatty acid composition. This study explores the biophysical effects of incorporating straight-chain unsaturated fatty acids into S. aureus membrane lipids. Membrane preparations from cultures supplemented with oleic acid showed more complex differential scanning calorimetry scans than those grown in tryptic soy broth alone. When grown in the presence of oleic acid, the cultures exhibited a transition significantly above the growth temperature, attributed to the presence of glycolipids with long-chain fatty acids causing acyl chain packing frustration within the bilayer. Functional aspects of the membrane were assessed by studying the kinetics of dye release from unilamellar vesicles induced by the antimicrobial peptide mastoparan X. Dye release was slower from liposomes prepared from cells grown in oleic acid-supplemented cultures, suggesting that changes in membrane lipid composition and biophysics protect the cell membrane against peptide-induced lysis. These findings underscore the intricate relationship between the growth environment, membrane lipid composition, and the physical properties of the bacterial membrane, which should be considered when developing new strategies against S. aureus infections.

biophysics↗

bioRxiv2024

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids over biosynthesized endogenous branched-chain fatty acids

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media, and when growing in vivo in an infection. Given the enhancement of membrane fluidity when oleic acid (C18:1{Delta}9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1{Delta}9 on growth at low temperatures. C18:1{Delta}9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12{degrees}C. Interestingly, we found similar results in the BCFA-sufficient parental strain. We show that incorporation of C18:1{Delta}9 and its elongation product C20:1{Delta}9 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol (PG) and diglycosyldiacylglycerol (DGDG) lipid classes revealed major impacts of C18:1{Delta}9 and temperature on lipid species. Growth at 12{degrees}C in the presence of C18:1{Delta}9 also led to increased production of the carotenoid pigment staphyloxanthin; however, this was not an obligatory requirement for cold adaptation. Enhancement of growth by C18:1{Delta}9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1{Delta}9 and other SCUFAs in various forms. IMPORTANCEWe show that S. aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerol bearing one or two degrees of unsaturation derived from incorporation of C18:1{Delta}9 at 12{degrees}C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying in membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance growth of S. aureus in the food environment.

microbiology↗

bioRxiv2023

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia

Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function can be mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include priming MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/567730v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1fca79forg.highwire.dtl.DTLVardef@60f543org.highwire.dtl.DTLVardef@186394borg.highwire.dtl.DTLVardef@11b94bd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMSCs morphologically respond to inflammatory priming conditions. C_LIO_LIPriming hits identified from morphological screen increase MSC-EV production. C_LIO_LIPriming MSCs in bioreactors enhances MSC-EV modulation of microglia. C_LIO_LIChanges in MSC-EV production and potency reflected by lipid content. C_LIO_LIFirst demonstration of effects of priming on MSC production of EVs in a bioreactor. C_LI

bioengineering↗

bioRxiv2023

Increased membrane fluidity and cell wall thickening contribute to high-level daptomycin resistance in S. aureus with defective pgsA and yycG

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin-resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin-resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. RT-qPCR studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCEThe cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to {beta}-lactams or vancomycin. Since daptomycins activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

microbiology↗

bioRxiv2022

Endocytosis in Trypanosoma cruzi Depends on Proper Recruitment and Regulation of Functionally Redundant Myosin Motors

Utilized by the free-living kinetoplastid Bodo saltans to feed on bacterial prey, the cytostome-cytopharynx complex (SPC) is an endocytic organelle absent from all human trypanosomatid pathogens save Trypanosoma cruzi. Building upon our previous work identifying the myosin motor MyoF as the first enzymatic component of the T. cruzi SPC, we sought to expand our understanding of this distinct organelle by identifying additional protein machinery which contribute to the endocytic process. While deletion of MyoF alone did not fully ablate endocytosis, we found that deletion of both MyoF and the similarly localized MyoC produced an endocytic-null phenotype that was rescued upon complementation. To identify potential regulatory components of this motor complex, we pulled down MyoF and identified an SPC-targeted protein that contained an annotated EF-hand calcium-binding motif that was conserved across a wide range of protozoan lineages. Surprisingly, deletion of this myosin associated protein (MyAP) alone was sufficient to produce an endocytic-null phenotype, which we were able to fully rescue via complementation. The deletion of MyAP also caused the mis-localization of both cytopharynx myosins to the cytosol. While MyAP lacking the EF-hand domain was unable to complement endocytosis, it was sufficient to restore proper myosin localization. This suggested that MyAP plays two distinct roles, one in targeting myosins to the SPC and a second in regulating myosin motor activity. Transmission electron microscopy also revealed that endocytic-null mutants lacked the electron lucent lipid inclusions typically seen in the pre-lysosomal reservosomes of T. cruzi epimastigotes. Mass spectrometry based lipidomic analysis subsequently revealed a dramatic reduction in the scavenged cholesterol content in the endocytic-null mutants, which can be attributed to an inability to endocytose exogenous lipid-protein complexes for storage in the reservosomes. Overall, this work showcases the first viable endocytic-null mutants generated in T. cruzi through specific gene deletion and highlights the feasibility of leveraging this strategy towards a full dissection of the endocytic machinery and biogenesis of the SPC. ImportanceTrypanosoma cruzi chronically infects over 7 million people in the Americas and current therapeutics are insufficient to effectively cure infection. The lack of progress in developing effective vaccines or drug treatments is due, in part, to longstanding technical limitations in studying this parasite and a lack of resources committed to support research and eradication efforts. As part of its parasitic lifestyle, T. cruzi is forced to obtain basic nutrients directly from its host environment, making the development of methods to block nutrient uptake an attractive strategy to control parasite growth and transmission. While the bulk uptake of complex nutrients by T. cruzi occurs via an endocytic structure, often referred to as the cytostome-cytopharynx complex (SPC), how exactly this tubular endocytic organelle functions at a mechanistic level has remained a mystery. In this work, we investigated the contribution of several SPC targeted myosin motors and an associated protein factor to endocytic activity. By identifying and characterizing the molecular machinery responsible for nutrient uptake, we hope to both expand our basic understanding of how this deadly pathogen acquires essential nutrients from its host, while also revealing new potential therapeutic targets to impede nutrient uptake.

microbiology↗

bioRxiv2021

7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome

Defective 3{beta}-hydroxysterol-{Delta}7 -reductase (DHCR7) in the developmental disorder, Smith-Lemli-Opitz syndrome (SLOS), results in deficiency in cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC). Here, we show that loss of DHCR7 causes accumulation of 7-DHC-derived oxysterol metabolites, premature neurogenesis, and perturbation of neuronal localization in developing murine or human cortical neural precursors, both in vitro and in vivo. We found that a major oxysterol, 3{beta},5-dihydroxycholest-7-en-6-one (DHCEO), mediates these effects by initiating crosstalk between glucocorticoid receptor (GR) and neurotrophin receptor kinase TrkB. Either loss of DHCR7 or direct exposure to DHCEO causes hyperactivation of GR and TrkB and their downstream MEK-ERK-C/EBP signaling pathway in cortical neural precursors. Moreover, direct inhibition of GR activation with an antagonist or inhibition of DHCEO accumulation with antioxidants rescues the premature neurogenesis phenotype caused by the loss of DHCR7. These results suggest that GR could be a new therapeutic target against the neurological defects observed in SLOS.

neuroscience↗

bioRxiv2020

Lipidomic and Ultrastructural Characterization of Cell Envelope of Staphylococcus aureus Grown in the Presence of Human Serum

Staphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained much fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus requires a living or non-denatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed CEs and TGs being the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host. IMPORTANCEComprehensive lipidomics of S. aureus grown in the presence of human serum suggests human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids-derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo. Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.

microbiology↗

bioRxiv2019

Occurrence of cross-resistance and beta-lactam seesaw effect in glycopeptide, lipopeptide, and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels

Treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections is challenging and is associated with high rates of therapeutic failure. The glycopeptide (GP) vancomycin and the lipopeptide (LP) daptomycin are still relied upon to manage invasive MRSA infections; however, resistance to these antibiotics has emerged and there is evidence of cross-resistance between them. It has been observed that the susceptibility of MRSA to beta-lactams increases as susceptibility to GPs and LPs decreases, a phenomenon termed the seesaw effect. Recent efforts to understand the mechanism underlying the seesaw effect have focused on the penicillin binding proteins (PBPs). However, while daptomycin resistance is largely mediated by remodeling of membrane lipid composition, the role of membrane lipids in producing cross-resistance and the seesaw effect has not yet been investigated. Here, we evaluate the lipid profiles, cross susceptibilities, and beta-lactam susceptibilities of a collection of isogenic MRSA strains selected against daptomycin, vancomycin or dalbavancin (a lipoglycopeptide; LGP) to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that typically the levels of short-chain phosphatidylglycerols (PGs) negatively correlate with MICs of GP/LP/LGP and positively correlate with MIC of certain beta-lactams, the latter being dependent on the primary PBP target of the particular beta-lactam. Furthermore, changes to certain PGs with long-chain fatty acids correlate well with presence of the seesaw effect. These studies demonstrate a major association between membrane remodeling and the seesaw effect.

microbiology↗

bioRxiv2019

A Vibrio cholerae BolA-like protein is required for proper cell shape and cell envelope integrity

BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well-described. Here, we investigated the role of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogens morphology. Notably, the bizarre-shaped, multi-polar, elongated and wide cells that predominated in exponential phase {Delta}ibaG V. cholerae cultures were not observed in stationary phase cultures. The V. cholerae {Delta}ibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall acting antibiotics and bile, and was defective in intestinal colonization. {Delta}ibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutants morphological defects and sensitivity to envelope stressors. Transposon-insertion sequencing analysis of ibaGs genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, co-purification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.\n\nImportanceBolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

microbiology↗