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To enhance the research capabilities of investigators interested in Staphylococcus aureus , the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen. IMPORTANCE Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website ( http://app1.unmc.edu/fgx/ ) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems. Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website ( http://app1.unmc.edu/fgx/ ) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems.

Recent studies have demonstrated a role for Staphylococcus aureus cidA-mediated cell lysis and genomic DNA release in biofilm adherence. The current study extends these findings by examining both temporal and additional genetic factors involved in the control of genomic DNA release and degradation during biofilm maturation. Cell lysis and DNA release were found to be critical for biofilm attachment during the initial stages of development and the released DNA (eDNA) remained an important matrix component during biofilm maturation. This study also revealed that an lrgAB mutant exhibits increased biofilm adherence and matrix-associated eDNA consistent with its proposed role as an inhibitor of cidA-mediated lysis. In flow-cell assays, both cid and lrg mutations had dramatic effects on biofilm maturation and tower formation. Finally, staphylococcal thermonuclease was shown to be involved in biofilm development as a nuc mutant formed a thicker biofilm containing increased levels of matrix-associated eDNA. Together, these findings suggest a model in which the opposing activities of the cid and lrg gene products control cell lysis and genomic DNA release during biofilm development, while staphylococcal thermonuclease functions to degrade the eDNA, possibly as a means to promote biofilm dispersal.

The Staphylococcus aureus cidABC and lrgAB operons represent the founding members of a large, highly conserved family of genes that span multiple kingdoms of life. Despite the fact that they have been shown to be involved in bacterial PCD during biofilm development, very little is known about the molecular/biochemical functions of the proteins they encode. The Staphylococcus aureus cidABC and lrgAB operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a “lysis cassette” system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. Typical of holins, CidA- and LrgA-induced lysis was dependent on the coexpression of endolysin, consistent with the proposed holin-like functions of these proteins. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence of their hole-forming potential. Consistent with recent reports demonstrating a role for the lrgAB homologues in other bacterial and plant species in the transport of by-products of carbohydrate metabolism, we also show that lrgAB is important for S. aureus to utilize pyruvate during microaerobic and anaerobic growth, by promoting the uptake of pyruvate under these conditions. Combined, these data reveal that the CidA and LrgA membrane proteins possess holin-like properties that play an important role in the transport of small by-products of carbohydrate metabolism. IMPORTANCE The Staphylococcus aureus cidABC and lrgAB operons represent the founding members of a large, highly conserved family of genes that span multiple kingdoms of life. Despite the fact that they have been shown to be involved in bacterial PCD, very little is known about the molecular/biochemical functions of the proteins they encode. The results presented in this study reveal that the cidA and lrgA genes encode proteins with bacteriophage holin-like functions, consistent with their roles in cell death. However, these studies also demonstrate that these operons are involved in the transport of small metabolic by-products of carbohydrate metabolism, suggesting an intriguing link between these two seemingly disparate processes.

Antimicrobial resistance (AMR) poses a global health threat, severely impeding the effective treatment of bacterial infections and jeopardizing the safety of routine medical procedures. Methicillin-resistant Staphylococcus aureus (MRSA) is particularly problematic because of its resistance to beta-lactams and the ability to form resilient biofilms. Conventional antibiotics, including last-resort options, have serious side effects and may contribute to further resistance. Chitosan, a natural biopolymer, offers a promising alternative due to its biocompatibility and antimicrobial properties, though its effectiveness against biofilms is limited. Recent studies suggest that increasing the positive charge density and adding hydrophobic moieties to chitosan, can enhance its antimicrobial properties. In this work, the antibacterial activity of quaternized chitosan derivatives against AMR S. aureus strains was assessed. Quaternization of chitosan’s amino group and introduction of hydrophobic side chains was found to significantly inhibit bacterial growth in both methicillin-sensitive (MSSA) and MRSA strains. Notably, nanofibrous materials composed of polyethylene oxide and hexyl-modified chitosan demonstrate alterations in S. aureus biofilm development, leading to significant accumulation of dead cells. Combined, these results highlight the potential of modified chitosan derivatives as effective antimicrobial agents for surface treatments and medical device coatings, particularly in applications where antibiotics are traditionally used, such as biofilm-prone environments.

The Staphylococcus aureus cidA and lrgA genes have been shown to affect cell lysis under a variety of conditions during planktonic growth. It is hypothesized that these genes encode holins and antiholins, respectively, and may serve as molecular control elements of bacterial cell lysis. To examine the biological role of cell death and lysis, we studied the impact of the cidA mutation on biofilm development. Interestingly, this mutation had a dramatic impact on biofilm morphology and adherence. The cidA mutant (KB1050) biofilm exhibited a rougher appearance compared with the parental strain (UAMS-1) and was less adherent. Propidium iodide staining revealed that KB1050 accumulated more dead cells within the biofilm population relative to UAMS-1, indicative of reduced cell lysis. In agreement with this finding, quantitative real-time PCR experiments demonstrated the presence of 5-fold less genomic DNA in the KB1050 biofilm relative to UAMS-1. Furthermore, treatment of the UAMS-1 biofilm with DNase I caused extensive cell detachment, whereas similar treatment of the KB1050 biofilm had only a modest effect. These results demonstrate that cidA-controlled cell lysis plays a significant role during biofilm development and that released genomic DNA is an important structural component of S. aureus biofilm.

Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC) and α-acetolactate synthase/decarboxylase (AlsSD) overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.

Staphylococcus aureus is an important human pathogen capable of colonizing diverse tissue types and inducing severe disease in both immunocompromised and otherwise healthy individuals. Biofilm infections caused by this bacterial species are of particular concern because of their persistence, even in the face of intensive therapeutic intervention. The results of the current study demonstrate the stochastic nature of Sae-mediated virulence gene expression in S. aureus and indicate that this regulatory system may function as a “bistable switch” in a manner similar to that seen with regulators controlling competence gene expression in Bacillus subtilis and persister cell formation in Escherichia coli . The results of this study provide a new perspective on the complex mechanisms utilized by S. aureus during the establishment of infections. The intricate process of biofilm formation in the human pathogen Staphylococcus aureus involves distinct stages during which a complex mixture of matrix molecules is produced and modified throughout the developmental cycle. Early in biofilm development, a subpopulation of cells detaches from its substrate in an event termed “exodus” that is mediated by SaePQRS-dependent stochastic expression of a secreted staphylococcal nuclease, which degrades extracellular DNA within the matrix, causing the release of cells and subsequently allowing for the formation of metabolically heterogenous microcolonies. Since the SaePQRS regulatory system is involved in the transcriptional control of multiple S. aureus virulence factors, the expression of several additional virulence genes was examined within a developing biofilm by introducing fluorescent gene reporter plasmids into wild-type S. aureus and isogenic regulatory mutants and growing these strains in a microfluidic system that supplies the bacteria with a constant flow of media while simultaneously imaging developing biofilms in 5-min intervals. This study demonstrated that multiple virulence genes, including nuc , were expressed stochastically within a specialized subpopulation of cells in nascent biofilms. We demonstrated that virulence genes regulated by SaePQRS were stochastically expressed in nearly all strains examined whereas Agr-regulated genes were expressed more homogenously within maturing microcolonies. The commonly used Newman strain contains a variant of SaeS (SaeS P ) that confers constitutive kinase activity to the protein and caused this strain to lack the stochastic expression pattern observed in other strain backgrounds. Importantly, repair of the SaeS P allele resulting in reversion to the well-conserved SaeS L allele found in other strains restored stochastic expression in this strain. IMPORTANCE Staphylococcus aureus is an important human pathogen capable of colonizing diverse tissue types and inducing severe disease in both immunocompromised and otherwise healthy individuals. Biofilm infections caused by this bacterial species are of particular concern because of their persistence, even in the face of intensive therapeutic intervention. The results of the current study demonstrate the stochastic nature of Sae-mediated virulence gene expression in S. aureus and indicate that this regulatory system may function as a “bistable switch” in a manner similar to that seen with regulators controlling competence gene expression in Bacillus subtilis and persister cell formation in Escherichia coli . The results of this study provide a new perspective on the complex mechanisms utilized by S. aureus during the establishment of infections.

Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC) and α-acetolactate synthase/decarboxylase (AlsSD) overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.

The Staphylococcus aureus cidA and lrgA genes have been shown to affect cell lysis under a variety of conditions during planktonic growth. It is hypothesized that these genes encode holins and antiholins, respectively, and may serve as molecular control elements of bacterial cell lysis. To examine the biological role of cell death and lysis, we studied the impact of the cidA mutation on biofilm development. Interestingly, this mutation had a dramatic impact on biofilm morphology and adherence. The cidA mutant (KB1050) biofilm exhibited a rougher appearance compared with the parental strain (UAMS-1) and was less adherent. Propidium iodide staining revealed that KB1050 accumulated more dead cells within the biofilm population relative to UAMS-1, indicative of reduced cell lysis. In agreement with this finding, quantitative real-time PCR experiments demonstrated the presence of 5-fold less genomic DNA in the KB1050 biofilm relative to UAMS-1. Furthermore, treatment of the UAMS-1 biofilm with DNase I caused extensive cell detachment, whereas similar treatment of the KB1050 biofilm had only a modest effect. These results demonstrate that cidA-controlled cell lysis plays a significant role during biofilm development and that released genomic DNA is an important structural component of S. aureus biofilm.

Imaging the brain distribution of translocator protein (TSPO), a putative biomarker for glial cell activation and neuroinflammation, may inform management of individuals infected with HIV by uncovering regional abnormalities related to neurocognitive deficits and enable non-invasive therapeutic monitoring. Using the second-generation TSPO-targeted radiotracer, [ 11 C]DPA-713, we conducted a positron emission tomography (PET) study to compare the brains of 12 healthy human subjects to those of 23 individuals with HIV who were effectively treated with combination antiretroviral therapy (cART). Compared to PET data from age-matched healthy control subjects, [ 11 C]DPA-713 PET of individuals infected with HIV demonstrated significantly higher volume-of-distribution (V T ) ratios in white matter, cingulate cortex, and supramarginal gyrus, relative to overall gray matter V T , suggesting localized glial cell activation in susceptible regions. Regional TSPO abnormalities were evident within a sub-cohort of neuro-asymptomatic HIV subjects, and an increase in the V T ratio within frontal cortex was specifically linked to individuals affected with HIV-associated dementia. These findings were enabled by employing a gray matter normalization approach for PET data quantification, which improved test–retest reproducibility, intra-class correlation within the healthy control cohort, and sensitivity of uncovering abnormal regional findings.