Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus-host cell interactions, shedding light on modulating host immune responses to S. aureus, which could involve developing immunomodulatory therapies. IMPORTANCE: This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Comprehensive virulence profiling and evolutionary analysis of specificity determinants in Staphylococcus aureus two-component systems.

In the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) and their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant methicillin-resistant S. aureus strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs' influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the co-crystallized structure of the DesK-DesR from Bacillus subtilis and the modeled structures of the four NarL TCSs in S. aureus, we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and the coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs.IMPORTANCEGiven the widespread conservation of two-component systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents, especially in the face of rising multi-drug-resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence, and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.

Comparative analysis of Vibrio cholerae isolates from Ghana reveals variations in genome architecture and adaptation of outbreak and environmental strains.

Recurrent epidemics of cholera denote robust adaptive mechanisms of Vibrio cholerae for ecological shifting and persistence despite variable stress conditions. Tracking the evolution of pathobiological traits requires comparative genomic studies of isolates from endemic areas. Here, we investigated the genetic differentiation among V. cholerae clinical and environmental isolates by highlighting the genomic divergence associated with gene decay, genome plasticity, and the acquisition of virulence and adaptive traits. The clinical isolates showed high phylogenetic relatedness due to a higher frequency of shared orthologs and fewer gene variants in contrast to the evolutionarily divergent environmental strains. Divergence of the environmental isolates is linked to extensive genomic rearrangements in regions containing mobile genetic elements resulting in numerous breakpoints, relocations, and insertions coupled with the loss of virulence determinants acf, zot, tcp, and ctx in the genomic islands. Also, four isolates possessed the CRISPR-Cas systems with spacers specific for Vibrio phages and plasmids. Genome synteny and homology analysis of the CRISPR-Cas systems suggest horizontal acquisition. The marked differences in the distribution of other phage and plasmid defense systems such as Zorya, DdmABC, DdmDE, and type-I Restriction Modification systems among the isolates indicated a higher propensity for plasmid or phage disseminated traits in the environmental isolates. Our results reveal that V. cholerae strains undergo extensive genomic rearrangements coupled with gene acquisition, reflecting their adaptation during ecological shifts and pathogenicity.

Nutrient Availability and Phage Exposure Alter the Quorum-Sensing and CRISPR-Cas-Controlled Population Dynamics of Pseudomonas aeruginosa.

Quorum sensing (QS) coordinates bacterial communication and cooperation essential for virulence and dominance in polymicrobial settings. QS also regulates the CRISPR-Cas system for targeted defense against parasitic genomes from phages and horizontal gene transfer. Although the QS and CRISPR-Cas systems are vital for bacterial survival, they undergo frequent selection in response to biotic and abiotic factors. Using the opportunistic Pseudomonas aeruginosa with well-established QS and CRISPR-Cas systems, we show how the social interactions between the acyl-homoserine lactone (AHL)-QS signal-blind mutants (ΔlasRrhlR) and the CRISPR-Cas mutants are affected by phage exposure and nutrient availability. We demonstrate that media conditions and phage exposure alter the resistance and relative fitness of ΔlasRrhlR and CRISPR-Cas mutants while tipping the fitness advantage in favor of the QS signal-blind mutants under nutrient-limiting conditions. We also show that the AHL signal-blind mutants are less selected by phages under QS-inducing conditions than the CRISPR-Cas mutants, whereas the mixed population of the CRISPR-Cas and AHL signal-blind mutants reduce phage infectivity, which can improve survival during phage exposure. Our data reveal that phage exposure and nutrient availability reshape the population dynamics between the ΔlasRrhlR QS mutants and CRISPR-Cas mutants, with key indications for cooperation and conflict between the strains. IMPORTANCE The increase in antimicrobial resistance has created the need for alternative interventions such as phage therapy. However, as previously observed with antimicrobial resistance, phage therapy will not be effective if bacteria evolve resistance and persist in the presence of the phages. The QS is commonly known as an arsenal for bacteria communication, virulence, and regulation of the phage defense mechanism, the CRISPR-Cas system. The QS and CRISPR-Cas systems are widespread in bacteria. However, they are known to evolve rapidly under the influence of biotic and abiotic factors in the bacterial environment, resulting in alteration in bacterial genotypes, which enhance phage resistance and fitness. We believe that adequate knowledge of the influence of environmental factors on the bacterial community lifestyle and phage defense mechanisms driven by the QS and CRISPR-Cas system is necessary for developing effective phage therapy.

Microevolution of the mexT and lasR Reinforces the Bias of Quorum Sensing System in Laboratory Strains of Pseudomonas aeruginosa PAO1.

The Pseudomonas aeruginosa strain PAO1 has routinely been used as a laboratory model for quorum sensing (QS). However, the microevolution of P. aeruginosa laboratory strains resulting in genetic and phenotypic variations have caused inconsistencies in QS research. To investigate the underlying causes of these variations, we analyzed 5 Pseudomonas aeruginosa PAO1 sublines from our laboratory using a combination of phenotypic characterization, high throughput genome sequencing, and bioinformatic analysis. The major phenotypic variations among the sublines spanned across the levels of QS signals and virulence factors such as pyocyanin and elastase. Furthermore, the sublines exhibited distinct variations in motility and biofilm formation. Most of the phenotypic variations were mapped to mutations in the lasR and mexT, which are key components of the QS circuit. By introducing these mutations in the subline PAO1-E, which is devoid of such mutations, we confirmed their influence on QS, virulence, motility, and biofilm formation. The findings further highlight a possible divergent regulatory mechanism between the LasR and MexT in the P. aeruginosa. The results of our study reveal the effects of microevolution on the reproducibility of most research data from QS studies and further highlight mexT as a key component of the QS circuit of P. aeruginosa.

Regulation of Exopolysaccharide Production by ProE, a Cyclic-Di-GMP Phosphodiesterase in Pseudomonas aeruginosa PAO1.

The ubiquitous second messenger c-di-GMP is involved in regulation of multiple biological functions including the important extracellular matrix exopolysaccharides (EPS). But how c-di-GMP metabolic proteins influence EPS and their enzymatic properties are not fully understood. Here we showed that deletion of proE, which encodes a protein with GGDEF-EAL hybrid domains, significantly increased the transcriptional expression of the genes encoding EPS production in Pseudomonas aeruginosa PAO1 and changed the bacterial colony morphology. Our data showed that ProE is a very active phosphodiesterase (PDE), with a high enzyme activity in degradation of c-di-GMP. Interestingly, the optimal activity of ProE was found in the presence of Co2+, unlike other PDEs that commonly rely on Mg2+ or Mn2+ for best performance. Furthermore, we identified three widely conserved novel residues that are critical for the function of ProE through site-directed mutagenesis. Subsequent study showed that ProE, together with other three key PDEs, i.e., RbdA, BifA, and DipA regulate the EPS production in P. aeruginosa PAO1. Moreover, by using the GFP-fusion approach, we observed that these four EPS associated-PDEs showed a polar localization pattern in general. Taken together, our data unveil the molecular mechanisms of ProE in regulation of EPS production, and provide a new insight on its enzymatic properties in degradation of c-di-GMP.

Molecular Mechanisms of Phosphate Stress Activation of Pseudomonas aeruginosa Quorum Sensing Systems.

The hierarchical quorum sensing (QS) systems of Pseudomonas aeruginosa, consisting of las, pqs, and rhl, coordinate the expression of bacterial virulence genes. Previous studies showed that under phosphate deficiency conditions, two-component regulatory system PhoRB could activate various genes involved in cytotoxicity through modulation of QS systems, but the mechanism by which PhoR/PhoB influences QS remains largely unknown. Here, we provide evidence that among the key QS regulatory genes in P. aeruginosa, rhlR, pqsA, mvfR, and lasI were activated by the response regulator PhoB under phosphate-depleted conditions. We show that PhoB is a strong competitor against LasR and RsaL for binding to the promoter of lasI and induces significant expression of lasI, rhlR, and mvfR. However, expression of lasI, encoding the signal 3-oxo-C12-HSL, was increased only marginally under the same phosphate-depleted conditions. This seeming inconsistency was attributed to the induction of pvdQ, which encodes an enzyme for degradation of 3-oxo-C12-HSL signal molecules. Taken together, the results from this study demonstrate that through the two-component regulatory system PhoR/PhoB, phosphate depletion stress could influence the QS network by modulating several key regulators, including lasI, rhlR, mvfR, and pvdQ The findings highlight not only the potency of the PhoR/PhoB-mediated bacterial stress response mechanism but also the plasticity of the P. aeruginosa QS systems in coping with the changed environmental conditions.IMPORTANCE It is not fully understood how phosphate deficiency could influence the virulence of Pseudomonas aeruginosa through modulation of the bacterial QS systems. This report presents a systemic investigation on the impact of phosphate depletion on the hierarchy of quorum sensing systems of P. aeruginosa The results showed that phosphate stress could have an extensive impact on the QS networks of this bacterial pathogen. Among the 7 QS regulatory genes representing the 3 sets of QS systems tested, 4 were significantly upregulated by phosphate depletion stress through the PhoR/PhoB two-component regulatory system, especially the upstream QS regulatory gene lasI We also present evidence that the response regulator PhoB was a strong competitor against the las regulators LasR and RsaL for the lasI promoter, unveiling the mechanistic basis of the process by which phosphate stress could modulate the bacterial QS systems.

Small Is Mighty-Chemical Communication Systems in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a variety of acute and chronic infections. Usually a commensal on the host body, P. aeruginosa is capable of transforming into a virulent pathogen upon sensing favorable changes in the host immune system or stress cues. P. aeruginosa infections are hard to eradicate, because this pathogen has developed strong resistance to most conventional antibiotics; in addition, in chronic infections it commonly forms a biofilm matrix, which provides bacterial cells a protected environment to withstand various stresses including antibiotics. Given its importance as a human pathogen and its notorious antimicrobial tolerance, P. aeruginosa has been the subject of intensive investigations internationally. Research progress over the last two decades has unveiled a range of chemical communication systems in this pathogen. These diversified chemical communication systems endow P. aeruginosa a superb ability and remarkable flexibility to coordinate and modulate accordingly the transcriptional expression of various sets of genes associated with virulence and other physiologic activities in response to environmental changes. A fair understanding of the chemical signaling mechanisms with which P. aeruginosa governs virulence gene expression may hold the key to developing alternative therapeutic interventions that control and prevent bacterial infections.