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1. Mechanisms and Implications of Bacterial Invasion across the Human Skin Barrier

   https://doi.org/10.1128/spectrum.02744-21  

   Lipsky, Zachary W.; Patsy, Marisa; Marques, Cláudia N.; German, Guy K. (June 2022, Microbiology Spectrum)

   Claesen, Jan (Ed.)

   ABSTRACT Atopic dermatitis (AD) is associated with a deficiency of skin lipids, increased populations of Staphylococcus aureus in the microbiome, and structural defects in the stratum corneum (SC), the outermost layer of human skin. However, the pathogenesis of AD is ambiguous, as it is unclear whether observed changes are the result of AD or contribute to the pathogenesis of the disease. Previous studies have shown that S. aureus is capable of permeating across isolated human SC tissue when lipids are depleted to levels consistent with AD conditions. In this study, we expand upon this discovery to determine the mechanisms and implications of bacterial penetration into the SC barrier. Specifically, we establish if bacteria are permeating intercellularly or employing a combination of both inter- and intracellular travel. The mechanical implications of bacterial invasion, lipid depletion, and media immersion are also evaluated using a newly developed, physiologically relevant, temperature-controlled drip chamber. Results reveal for the first time that S. aureus can be internalized by corneocytes, indicating transcellular movement through the tissue during permeation, consistent with previous theoretical models. S. aureus also degrades the mechanical integrity of human SC, particularly when the tissue is partially depleted of lipids. These observed mechanical changes are likely the cause of broken or ruptured tissue seen as exudative lesions in AD flares. This work further highlights the necessity of lipids in skin microbial barrier function. IMPORTANCE Millions of people suffer from the chronic inflammatory skin disease atopic dermatitis (AD), whose symptoms are associated with a deficiency of skin lipids that exhibit antimicrobial functions and increased populations of the opportunistic pathogen Staphylococcus aureus . However, the pathogenesis of AD is ambiguous, and it remains unclear if these observed changes are merely the result of AD or contribute to the pathogenesis of the disease. In this article, we demonstrate the necessity of skin lipids in preventing S. aureus from penetrating the outermost barrier of human skin, thereby causing a degradation in tissue integrity. This bacterial permeation into the viable epidermis could act as an inflammatory trigger of the disease. When coupled with delipidated AD tissue conditions, bacterial permeation can also explain increased tissue fragility, potentially causing lesion formation in AD patients that results in further enhancing bacterial permeability across the stratum corneum and the development of chronic conditions. 

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2. Exogenous Fatty Acids Remodel Staphylococcus aureus Lipid Composition through Fatty Acid Kinase

   https://doi.org/10.1128/JB.00128-20  

   DeMars, Zachary; Singh, Vineet K.; Bose, Jeffrey L. (June 2020, Journal of Bacteriology)

   Stock, Ann M. (Ed.)

   ABSTRACT Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA . We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology. IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus . Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function. 

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3. Responses to chemical cross-talk between the Mycobacterium ulcerans toxin, mycolactone, and Staphylococcus aureus

   https://doi.org/10.1038/s41598-021-89177-5  

   Dhungel, Laxmi; Burcham, Lindsey; Park, Joo Youn; Sampathkumar, Harshini Devi; Cudjoe, Albert; Seo, Keun Seok; Jordan, Heather (December 2021, Scientific Reports)

   null (Ed.)

   Abstract Buruli ulcer is a neglected tropical disease caused by the environmental pathogen, Mycobacterium ulcerans whose major virulence factor is mycolactone, a lipid cytotoxic molecule. Buruli ulcer has high morbidity, particularly in rural West Africa where the disease is endemic. Data have shown that infected lesions of Buruli ulcer patients can be colonized by quorum sensing bacteria such as Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa , but without typical pathology associated with those pathogens’ colonization. M. ulcerans pathogenesis may not only be an individual act but may also be dependent on synergistic or antagonistic mechanisms within a polymicrobial network. Furthermore, co-colonization by these pathogens may promote delayed wound healing, especially after the initiation of antibiotic therapy. Hence, it is important to understand the interaction of M. ulcerans with other bacteria encountered during skin infection. We added mycolactone to S. aureus and incubated for 3, 6 and 24 h. At each timepoint, S. aureus growth and hemolytic activity was measured, and RNA was isolated to measure virulence gene expression through qPCR and RNASeq analyses. Results showed that mycolactone reduced S. aureus hemolytic activity, suppressed hla promoter activity, and attenuated virulence genes, but did not affect S. aureus growth . RNASeq data showed mycolactone greatly impacted S. aureus metabolism. These data are relevant and significant as mycolactone and S. aureus sensing and response at the transcriptional, translational and regulation levels will provide insight into biological mechanisms of interspecific interactions that may play a role in regulation of responses such as effects between M. ulcerans , mycolactone, and S. aureus virulence that will be useful for treatment and prevention. 

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4. Chromosome segregation dynamics during the cell cycle of Staphylococcus aureus

   https://doi.org/10.1101/2025.02.18.638847  

   Izquierdo-Martinez, Adrian; Schäper, Simon; Brito, António D; Liao, Qin; Tesseur, Coralie; Sorg, Moritz; Botinas, Daniela S; Wang, Xindan; Pinho, Mariana G (February 2025, bioRxiv)

   Abstract Research on chromosome organization and cell cycle progression in spherical bacteria, particularlyStaphylococcus aureus, remains limited and fragmented. In this study, we established a working model to investigate chromosome dynamics inS. aureususing a Fluorescent Repressor-Operator System (FROS), which enabled precise localization of specific chromosomal loci. This approach revealed that theS. aureuscell cycle and chromosome replication cycle are not coupled, with cells exhibiting two segregated origins of replication at the start of the cell cycle. The chromosome has a specific origin-terminus-origin conformation, with origins localizing near the membrane, towards the tip of each hemisphere, or the “cell poles”. We further used this system to assess the role of various proteins with a role inS. aureuschromosome biology, focusing on the ParB-parSand SMC-ScpAB systems. Our results demonstrate that ParB binds fiveparSchromosomal sequences and the resulting complexes influence chromosome conformation, but play a minor role in chromosome compaction and segregation. In contrast, the SMC-ScpAB complex plays a key role inS. aureuschromosome biology, contributing to chromosome compaction, segregation and spatial organization. Additionally, we systematically assessed and compared the impact of proteins linking chromosome segregation to cell division—Noc, FtsK, SpoIIIE and XerC—on origin and terminus number and positioning. This work provides a comprehensive study of the factors governing chromosome dynamics and organization inS. aureus, contributing to our knowledge on chromosome biology of spherical bacteria. 

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5. Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers

   https://doi.org/10.1016/j.jmsacl.2022.09.003  

   Rivera, Emilio S.; Weiss, Andy; Migas, Lukasz G.; Freiberg, Jeffrey A.; Djambazova, Katerina V.; Neumann, Elizabeth K.; Van de Plas, Raf; Spraggins, Jeffrey M.; Skaar, Eric P.; Caprioli, Richard M. (November 2022, Journal of Mass Spectrometry and Advances in the Clinical Lab)

   Introduction Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results Implementation of TIMS led to a ∼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) – CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies. 

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