Kelly Marie Hines

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. IMPORTANCE: S. 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.

Previous work suggests that Enterococcus faecalis uses exogenous oleic acid (FA 18:1(9z)) to increase its tolerance against the membrane-targeting antimicrobial, daptomycin. However, the specific roles of OA and the endogenous positional isomer, cis-vaccenic acid (FA 18:1(11z)), in both daptomycin-susceptible (Dap-S) and daptomycin-resistant (Dap-R) strains have not been fully explored. We performed lipidomics using hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography-ion mobility-mass spectrometry to identify alterations in the lipid composition of Dap-S (S613) and Dap-R (R712) strain pairs of E. faecalis following OA or CV supplementation. Lipidomics showed that total PG intensity in Dap-S was only impacted by OA, while total DGDG intensity was impacted by both OA and CV. However, total PG and DGDG intensities in Dap-R were not impacted by either FA. Both strains produced significantly more lipids containing two 18:1 acyl tails following OA supplementation, while CV had a similar effect on Dap-S only, indicating that Dap-S and Dap-R respond to OA and CV differently. Additionally, the preferences for OA vs CV, alone and in mixtures, were distinguished using ozonolysis and deuterium-labeling techniques that can resolve C═C positional isomers. Growth and survival assays show that Dap-S responds differently to daptomycin when it is cultured with OA or CV. Together, these results reveal that E. faecalis strains with genetic determinants for daptomycin resistance respond differently, in membrane lipid composition and overall growth, to biologically relevant FA 18:1 isomers compared to strains that acquire daptomycin tolerance solely through exogenous fatty acid uptake.

To maintain its barrier function, human skin requires lipids, including cholesterol, ceramides, free fatty acids, and wax esters. In contrast to other skin lipids, wax esters remain largely unexplored as metabolites of the skin microbiome. Recently, we discovered that Staphylococcus aureus utilises its lipase Lip2 to detoxify antimicrobial fatty acids (AFAs) by esterification with cholesterol. The observed promiscuity of Lip2 and the presence of similar lipases among skin staphylococci prompted the search for novel substrates and products. Here, we identify S. aureus Lip2 and Lip1, Staphylococcus simulans SsL, and Staphylococcus epidermidis GehD and GehC as lipases capable of metabolizing wax esters. These lipases degraded wax esters into AFAs and fatty alcohols, and, except for GehC, shared the ability to esterify AFAs with fatty alcohols, thereby generating wax esters. In monocultures of bacteria heterologously expressing staphylococcal lipases, synthesis of wax esters mirrored protection from AFAs by fatty alcohols in planktonic or biofilm settings. In pairwise cocultures, lipases secreted by lipase-proficient S. aureus, S. simulans, and S. epidermidis functioned as public goods, rescuing lipase-deficient mutants. In the absence of detoxifying substrates/lipases, S. simulans or S. epidermidis outcompeted S. aureus when exposed to AFAs. A skin-mimicking high-salt environment increased the resistance of S. aureus, but not S. simulans, to AFAs, enabling S. aureus to match or outcompete S. simulans in the presence of AFAs or AFAs plus fatty alcohols, respectively. Collectively, our findings suggest that commensal modulation of skin lipids determines whether niche-specific communities are resilient or permissive to pathogenic invasion by S. 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 show that 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, alterations to BCFA abundance impair S. aureus growth, compromise membrane integrity, diminish expression of the accessory gene regulator quorum-sensing system, and increase sensitivity to membrane-targeting antimicrobials. Disrupted BCFA metabolism also reduced S. aureus fitness in competition with Pseudomonas aeruginosa , suggesting that airway microbiome composition and the metabolites they exchange can directly impact pathogen succession over time. The pleiotropic effects of SCFAs on S. aureus fitness and their ubiquity as metabolites in the human host also suggest that they may be effective as adjuvants to traditional antimicrobial agents when used in combination. IMPORTANCE: Staphylococcus aureus is a primary pathogen of chronic airway disease yet is also found in the upper airways of 30%–50% of the population to no obvious detriment. Thus, identifying the host and/or microbial factors that tip the balance between its commensal and pathogenic states may be key to its control. Here, we reveal that short-chain fatty acids produced by commensal microbiota promote a marked remodeling of the S. aureus lipid membrane that, in turn, sensitizes the pathogen to antimicrobials, disrupts accessory gene regulator quorum signaling, and reduces its competitive fitness. Altogether, these data suggest that co-colonizing microbiota and the metabolites they exchange with S. aureus may be key players in the microbial ecology of airway disease.

The implementation of mass spectrometry (MS) in clinical microbiology has made a significant improvement in the turnaround time from positive culture to identification, but current protein-based approaches can struggle with species-level identification because of the high degree of homology within a genus. However, other MS-based strategies for bacterial identification that are based on lipids and small molecules have shown promise toward species-level identification and detection of specific phenotypes, including those related to antibiotic resistance. We are leveraging rapid gas-phase ion mobility (IM) separations coupled to MS to simultaneously detect the lipids and metabolites in bacterial pathogens. Using flow-injection (FI) rather than liquid chromatography (LC), we instead rely more directly on the structural separation of the IM dimension to resolve features from different biochemical classes and aid in identification. A head-to-head comparison demonstrates that the FI-IM-MS multiomic strategy performs similarly to LC-IM-MS in its ability to distinguish 24 strains of the high-concern ESKAPE pathogens, while shortening overall analysis time from 17 to 2 min per injection. We demonstrate that the IM dimension has excellent stability and reproducibility, which enables extracted IM peak areas to be used in lieu of chromatographic peak areas. Furthermore, the same features that are important for the discrimination of bacterial species and strains are found within both the FI-IM-MS and HILIC-IM-MS data sets. These results showcase the capabilities of mobility-enabled rapid multiomics and open the possibility to detect subtle strain-level differences and resistance phenotypes in bacterial pathogens by including additional classes of biomolecules.

The implementation of mass spectrometry (MS) in clinical microbiology has made significant improvement in the turnaround time from positive culture to identification, but current protein-based approaches can struggle with species level identifications because of the high degree of homology within a genus. However, other MS-based strategies for bacteria identifications that are based on lipids and small molecules have shown promise towards species-level identifications and detection of specific phenotypes, including those related to antibiotic resistance. While the concept of using multi-omics in diagnostic situations is not new, the issues of time and efficiency remain major hurdles to implementing multi-omics into situations that demand rapid and high-throughput analyses like clinical microbiology. We are addressing this gap by leveraging rapid, gas-phase ion mobility (IM) separations coupled to MS to simultaneously detect the lipids and metabolites in bacterial pathogens. Using flow-injection (FI) rather than liquid chromatography (LC), we instead rely more directly on the structural separations of the IM dimension to resolve features from different biochemical classes and aid in identifications. A head-to-head comparison demonstrates that the FI-IM-MS multi-omic strategy performs similarly to LC-IM-MS in its ability to distinguish 24 strains of the high-concern ESKAPE pathogens, while shortening overall analysis time from 17 min to 2 min per injection. We demonstrate that the IM dimension has excellent stability and reproducibility, which enables extracted IM peak areas to be used in lieu of chromatographic peak areas. Although the total number of features detected in the FI-IM-MS dataset is lower than the HILIC-IM-MS dataset, the overlap between the two datasets includes the features that are most heavily weighted in the PCA separation of the 24 strains. These results showcase the capabilities of mobility-enabled rapid multi-omics and open the possibility to detect subtle strain-level differences and resistance phenotypes in bacteria pathogens by including additional classes of biomolecules.

Campylobacter jejuni is a common foodborne pathogen worldwide that is associated with high rates of morbidity and mortality among infants in low- to middle-income countries (LMICs). Human milk provides infants with an important source of nutrients and contains antimicrobial components for protection against infection. However, recent studies, including our own, have found significantly higher levels of Campylobacter in diarrheal stool samples collected from breastfed infants compared to non-breastfed infants in LMICs. We hypothesized that C. jejuni has unique strategies to resist the antimicrobial properties of human milk. Transcriptional profiling found human milk exposure induces genes associated with ribosomal function, iron acquisition, and amino acid utilization in C. jejuni strains 81–176 and 11168. However, unidentified proteinaceous components of human milk prevent bacterial growth. Evolving both C. jejuni isolates to survive in human milk resulted in mutations in genes encoding the acyl carrier protein (AcpP) and the major outer membrane porin (PorA). Introduction of the PorA/AcpP amino acid changes into the parental backgrounds followed by electron microscopy showed distinct membrane architectures, and the AcpP changes not only significantly improved growth in human milk, but also yielded cells surrounded with outer membrane vesicles. Analyses of the phospholipid and lipooligosaccharide (LOS) compositions suggest an imbalance in acyl chain distributions. For strain 11168, these changes protect both evolved and 11168∆ acpP ^G33R strains from bacteriophage infection and polymyxin killing. Taken together, this study provides insights into how C. jejuni may evolve to resist the bactericidal activity of human milk and flourish in the hostile environment of the gastrointestinal tract. IMPORTANCE: In this study, we evolved C. jejuni strains which can grow in the presence of human milk and found that cell membrane alterations may be involved in resistance to the antimicrobial properties of human milk. These bacterial membrane changes are predominantly linked to amino acid substitutions within the acyl carrier protein, AcpP, although other bacterial components, including PorA, are likely involved. This study provides some insights into possible strategies for C. jejuni survival and propagation in the gastrointestinal tract of breastfed infants.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes significant global morbidity, including fever, rash, and persistent arthralgia. Utilizing untargeted lipidomics, we investigated how CHIKV infection alters host cell lipid metabolism in Vero cells. CHIKV infection induced marked catabolism of hexosylceramides, reducing their levels while increasing ceramide byproducts. Functional studies revealed a reliance on fatty acid synthesis, β-oxidation, and glycosphingolipid biosynthesis. Notably, inhibition of uridine diphosphate glycosyltransferase 8 (UGT8), essential for galactosylceramide production, significantly impaired CHIKV replication and entry in Vero cells. Sensitivity of CHIKV to UGT8 inhibition was reproduced in a disease-relevant cell line, mouse hepatocytes (Hepa1-6). CHIKV was also sensitive to evacetrapib, a cholesterol ester transfer protein (CETP) inhibitor, though the mechanism of inhibition appeared independent of CETP itself, suggesting an off-target effect. These findings highlight specific lipid pathways, particularly glycosphingolipid metabolism, as critical for CHIKV replication and further refine our understanding of how CHIKV exploits host lipid networks. This study provides new insights into CHIKV biology and suggests that targeted investigation of host lipid pathways may inform future therapeutic strategies.