Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

Tensor-based insights into systems immunity and infectious disease.

Profiling immune responses across several dimensions, including time, patients, molecular features, and tissue sites, can deepen our understanding of immunity as an integrated system. These studies require new analytical approaches to realize their full potential. We highlight recent applications of tensor methods and discuss several future opportunities.

PACAP is a pathogen-inducible resident antimicrobial neuropeptide affording rapid and contextual molecular host defense of the brain.

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

MRSA Isolates from Patients with Persistent Bacteremia Generate Nonstable Small Colony Variants In Vitro within Macrophages and Endothelial Cells during Prolonged Vancomycin Exposure.

Staphylococcus aureus (especially methicillin-resistant S. aureus [MRSA]) is frequently associated with persistent bacteremia (PB) during vancomycin therapy despite consistent susceptibility in vitro. Strategic comparisons of PB strains versus those from vancomycin-resolving bacteremia (RB) would yield important mechanistic insights into PB outcomes. Clinical PB versus RB isolates were assessed in vitro for intracellular replication and small colony variant (SCV) formation within macrophages and endothelial cells (ECs) in the presence or absence of exogenous vancomycin. In both macrophages and ECs, PB and RB isolates replicated within lysosome-associated membrane protein-1 (LAMP-1)-positive compartments. PB isolates formed nonstable small colony variants (nsSCVs) in vancomycin-exposed host cells at a significantly higher frequency than matched RB isolates (in granulocyte-macrophage colony-stimulating factor [GM-CSF], human macrophages PB versus RB, P < 0.0001 at 48 h; in ECs, PB versus RB, P < 0.0001 at 24 h). This phenotype could represent one potential basis for the unique ability of PB isolates to adaptively resist vancomycin therapy and cause PB in humans. Elucidating the molecular mechanism(s) by which PB strains form nsSCVs could facilitate the discovery of novel treatment strategies to mitigate PB due to MRSA.

Unifying structural signature of eukaryotic α-helical host defense peptides.

Diversity of α-helical host defense peptides (αHDPs) contributes to immunity against a broad spectrum of pathogens via multiple functions. Thus, resolving common structure-function relationships among αHDPs is inherently difficult, even for artificial-intelligence-based methods that seek multifactorial trends rather than foundational principles. Here, bioinformatic and pattern recognition methods were applied to identify a unifying signature of eukaryotic αHDPs derived from amino acid sequence, biochemical, and three-dimensional properties of known αHDPs. The signature formula contains a helical domain of 12 residues with a mean hydrophobic moment of 0.50 and favoring aliphatic over aromatic hydrophobes in 18-aa windows of peptides or proteins matching its semantic definition. The holistic α-core signature subsumes existing physicochemical properties of αHDPs, and converged strongly with predictions of an independent machine-learning-based classifier recognizing sequences inducing negative Gaussian curvature in target membranes. Queries using the α-core formula identified 93% of all annotated αHDPs in proteomic databases and retrieved all major αHDP families. Synthesis and antimicrobial assays confirmed efficacies of predicted sequences having no previously known antimicrobial activity. The unifying α-core signature establishes a foundational framework for discovering and understanding αHDPs encompassing diverse structural and mechanistic variations, and affords possibilities for deterministic design of antiinfectives.

Genetic variation of DNA methyltransferase-3A contributes to protection against persistent MRSA bacteremia in patients.

The role of the host in development of persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is not well understood. A cohort of prospectively enrolled patients with persistent methicillin-resistant S. aureus bacteremia (PB) and resolving methicillin-resistant S. aureus bacteremia (RB) matched by sex, age, race, hemodialysis status, diabetes mellitus, and presence of implantable medical device was studied to gain insights into this question. One heterozygous g.25498283A > C polymorphism located in the DNMT3A intronic region of chromosome 2p with no impact in messenger RNA (mRNA) expression was more common in RB (21 of 34, 61.8%) than PB (3 of 34, 8.8%) patients (P = 7.8 × 10-6). Patients with MRSA bacteremia and g.25498283A > C genotype exhibited significantly higher levels of methylation in gene-regulatory CpG island regions (Δmethylation = 4.1%, P < 0.0001) and significantly lower serum levels of interleukin-10 (IL-10) than patients with MRSA bacteremia without DNMT3A mutation (A/C: 9.7038 pg/mL vs. A/A: 52.9898 pg/mL; P = 0.0042). Expression of DNMT3A was significantly suppressed in patients with S. aureus bacteremia and in S. aureus-challenged primary human macrophages. Small interfering RNA (siRNA) silencing of DNMT3A expression in human macrophages caused increased IL-10 response upon S. aureus stimulation. Treating macrophages with methylation inhibitor 5-Aza-2'-deoxycytidine resulted in increased levels of IL-10 when challenged with S. aureus In the murine sepsis model, methylation inhibition increased susceptibility to S. aureus These findings indicate that g.25498283A > C genotype within DNMT3A contributes to increased capacity to resolve MRSA bacteremia, potentially through a mechanism involving increased methylation of gene-regulatory regions and reduced levels of antiinflammatory cytokine IL-10.

Protective immunity in recurrent Staphylococcus aureus infection reflects localized immune signatures and macrophage-conferred memory.

Staphylococcus aureus is the leading cause of skin and skin structure infection (SSSI), a primary portal of entry for invasive infection. Our prior studies discovered a role for protective innate memory against recurrent methicillin-resistant S. aureus (MRSA) SSSI. In the present study, the dynamics and mechanisms of this response were explored in recurrent SSSI in WT mice. Priming by prior infection reduced skin lesion severity and MRSA burden, and protected against dissemination at day 7 but not day 2. Cytokine and cellular signatures in SSSI differed at day 2 versus 7, and were distinct in skin versus blood or spleen. Cytokines associated with protection in skin included increased IL-17, IL-6, monokine inducible by IFN-γ (MIG), and RANTES, while increased IP-10 correlated with protection from dissemination. Cellular signatures of protection included increased Th17, M1 macrophage, and dendritic cell populations in abscesses, and total macrophages in lymph nodes. Priming potentiated S. aureus-specific phagocytic killing by bone marrow-derived macrophages in vitro, and their adoptive transfer into naïve skin afforded protective efficacy in vivo. Present findings indicate that protective immunity in recurrent S. aureus infection is locally targeted, and involves specific memory conferred by macrophages. These insights provide targets for vaccine and immunotherapeutic development against MRSA.

Innate Immune Memory Contributes to Host Defense against Recurrent Skin and Skin Structure Infections Caused by Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI). The high frequency of recurring SSSI due to S. aureus, including methicillin-resistant S. aureus (MRSA) strains, despite high titers of specific antibodies and circulating T cells, implies that traditional adaptive immunity imparts incomplete protection. We hypothesized that innate immune memory contributes to the protective host defense against recurring MRSA infection. To test this hypothesis, SSSI was induced in wild-type and rag1-/- mice in the BALB/c and C57BL/6 backgrounds. Prior infection (priming) of wild-type and rag1-/- mice of either background afforded protection against repeat infection, as evidenced by reduced abscess severities and decreased CFU densities compared to those in naive controls. Interestingly, protection was greater on the previously infected flank than on the naive flank for wild-type and rag1-/- mice. For wild-type mice, protective efficacy corresponded to increased infiltration of neutrophils (polymorphonuclear leukocytes [PMN]), macrophages (MΦ), Langerin+ dendritic cells (LDC), and natural killer (NK) cells. Protection was associated with the induction of interleukin-17A (IL-17A), IL-22, and gamma interferon (IFN-γ) as well as the antimicrobial peptides CRAMP and mßD-3. Priming also protected rag1-/- mice against recurring SSSI, with increased MΦ and LDC infiltration and induction of IL-22, CRAMP, and mßD-3. These findings suggest that innate immune memory, mediated by specific cellular and molecular programs, likely contributes to the localized host defense in recurrent MRSA SSSI. These insights support the development of targeted immunotherapeutic strategies to address the challenge of MRSA infection.

PBP 4 Mediates High-Level Resistance to New-Generation Cephalosporins in Staphylococcus aureus.

Staphylococcus aureus is an important cause of both hospital- and community-associated methicillin-resistant S. aureus (MRSA) infections worldwide. ß-Lactam antibiotics are the drugs of choice to treat S. aureus infections, but resistance to these and other antibiotics make treatment problematic. High-level ß-lactam resistance of S. aureus has always been attributed to the horizontally acquired penicillin binding protein 2a (PBP 2a) encoded by the mecA gene. Here, we show that S. aureus can also express high-level resistance to ß-lactams, including new-generation broad-spectrum cephalosporins that are active against methicillin-resistant strains, through an uncanonical core genome-encoded penicillin binding protein, PBP 4, a nonessential enzyme previously considered not to be important for staphylococcal ß-lactam resistance. Our results show that PBP 4 can mediate high-level resistance to ß-lactams.

Nonredundant Roles of Interleukin-17A (IL-17A) and IL-22 in Murine Host Defense against Cutaneous and Hematogenous Infection Due to Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.

Comparative efficacies of tedizolid phosphate, vancomycin, and daptomycin in a rabbit model of methicillin-resistant Staphylococcus aureus endocarditis.

Tedizolid, the active component of the prodrug tedizolid phosphate, is a novel oxazolidinone that is approximately 4 times more active by weight than linezolid against Staphylococcus aureus in vitro. The in vivo efficacy of tedizolid phosphate (15 mg/kg body weight intravenous [i.v.] twice a day [b.i.d.]) was compared to those of vancomycin (30 mg/kg i.v. b.i.d.) and daptomycin (18 mg/kg i.v. once a day [q.d.]) in a rabbit model of aortic valve endocarditis (AVE) caused by methicillin-resistant S. aureus strain COL (infection inoculum of 10(7) CFU). Median vegetation titers of daptomycin-treated rabbits were significantly lower than those of rabbits treated with tedizolid phosphate (15 mg/kg b.i.d.) (P = 0.016), whereas titers for vancomycin-treated compared to tedizolid-treated rabbits were not different (P = 0.984). The numbers of organisms in spleen and kidney tissues were similar for all treatment groups. A dose-ranging experiment was performed with tedizolid phosphate (2, 4, and 8 mg/kg b.i.d.) compared to vancomycin (30 mg/kg b.i.d.), using a higher infecting inoculum (10(8) CFU) to determine the lowest efficacious dose of tedizolid phosphate. Tedizolid phosphate (2 mg/kg) (equivalent to 60% of the area under the concentration-time curve from 0 to 24 h (AUC0-24) for the human 200-mg dose approved by the U.S. Food and Drug Administration) was not efficacious. Tedizolid phosphate at 4 mg/kg (equivalent to 75% of the AUC0-24 for the human 400-mg dose) and 8 mg/kg produced lower vegetation titers than the control, but neither was as efficacious as vancomycin.

Ceftobiprole- and ceftaroline-resistant methicillin-resistant Staphylococcus aureus.

The role of mecA mutations in conferring resistance to ceftobiprole and ceftaroline, cephalosporins with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, was determined with MRSA strains COL and SF8300. The SF8300 ceftaroline-passaged mutant carried a single mecA mutation, E447K (E-to-K change at position 447), and expressed low-level resistance. This mutation in COL conferred high-level resistance to ceftobiprole but only low-level resistance to ceftaroline. The COL ceftaroline-passaged mutant, which expressed high-level resistance to ceftobiprole and ceftaroline, had mutations in pbp2, pbp4, and gdpP but not mecA.

Integrated transcriptomic analysis reveals immune signatures distinguishing persistent versus resolving outcomes in MRSA bacteremia.

Introduction: Staphylococcus aureus bacteremia (SAB) is a life-threatening infection particularly involving methicillin-resistant S. aureus (MRSA). In contrast to resolving MRSA bacteremia (RB), persistent MRSA bacteremia (PB) blood cultures remain positive despite appropriate antibiotic treatment. Host immune responses distinguishing PB vs. RB outcomes are poorly understood. Here, integrated transcriptomic, IL-10 cytokine levels, and genomic analyses sought to identify signatures differentiating PB vs. RB outcomes. Methods: Whole-blood transcriptomes of propensity-matched PB (n=28) versus RB (n=30) patients treated with vancomycin were compared in one independent training patient cohort. Gene expression (GE) modules were analyzed and prioritized relative to host IL-10 cytokine levels and DNA methyltransferase-3A (DNMT3A) genotype. Results: Differential expression of T and B lymphocyte gene expression early in MRSA bacteremia discriminated RB from PB outcomes. Significant increases in effector T and B cell signaling pathways correlated with RB, lower IL-10 cytokine levels and DNMT3A heterozygous A/C genotype. Importantly, a second PB and RB patient cohort analyzed in a masked manner demonstrated high predictive accuracy of differential signatures. Discussion: Collectively, the present findings indicate that human PB involves dysregulated immunity characterized by impaired T and B cell responses associated with excessive IL-10 expression in context of the DNMT3A A/A genotype. These findings reveal distinct immunologic programs in PB vs. RB outcomes, enable future studies to define mechanisms by which host and/or pathogen drive differential signatures and may accelerate prediction of PB outcomes. Such prognostic assessment of host risk could significantly enhance early anti-infective interventions to avert PB and improve patient outcomes.

Tensor modeling of MRSA bacteremia cytokine and transcriptional patterns reveals coordinated, outcome-associated immunological programs.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a common and life-threatening infection that imposes up to 30% mortality even when appropriate therapy is used. Despite in vitro efficacy determined by minimum inhibitory concentration breakpoints, antibiotics often fail to resolve these infections in vivo, resulting in persistent MRSA bacteremia. Recently, several genetic, epigenetic, and proteomic correlates of persistent outcomes have been identified. However, the extent to which single variables or their composite patterns operate as independent predictors of outcome or reflect shared underlying mechanisms of persistence is unknown. To explore this question, we employed a tensor-based integration of host transcriptional and cytokine datasets across a well-characterized cohort of patients with persistent or resolving MRSA bacteremia outcomes. This method yielded high correlative accuracy with outcomes and immunologic signatures united by transcriptomic and cytokine datasets. Results reveal that patients with persistent MRSA bacteremia (PB) exhibit signals of granulocyte dysfunction, suppressed antigen presentation, and deviated lymphocyte polarization. In contrast, patients with resolving bacteremia (RB) heterogeneously exhibit correlates of robust antigen-presenting cell trafficking and enhanced neutrophil maturation corresponding to appropriate T lymphocyte polarization and B lymphocyte response. These results suggest that transcriptional and cytokine correlates of PB vs. RB outcomes are complex and may not be disclosed by conventional modeling. In this respect, a tensor-based integration approach may help to reveal consensus molecular and cellular mechanisms and their biological interpretation.

Diflunisal and Analogue Pharmacophores Mediating Suppression of Virulence Phenotypes in Staphylococcus aureus.

Invasive methicillin-resistant Staphylococcus aureus (MRSA) infections are leading causes of morbidity and mortality that are complicated by increasing resistance to conventional antibiotics. Thus, minimizing virulence and enhancing antibiotic efficacy against MRSA is a public health imperative. We originally demonstrated that diflunisal (DIF; [2-hydroxy-5-(2,4-difluorophenyl) benzoic acid]) inhibits S. aureus virulence factor expression. To investigate pharmacophores that are active in this function, we evaluated a library of structural analogues for their efficacy to modulate virulence phenotypes in a panel of clinically relevant S. aureus isolates in vitro. Overall, the positions of the phenyl, hydroxyl, and carboxylic moieties and the presence or type of halogen (F vs. Cl) influenced the efficacy of compounds in suppressing hemolysis, proteolysis, and biofilm virulence phenotypes. Analogues lacking halogens inhibited proteolysis to an extent similar to DIF but were ineffective at reducing hemolysis or biofilm production. In contrast, most analogues lacking the hydroxyl or carboxylic acid groups did not suppress proteolysis but did mitigate hemolysis and biofilm production to an extent similar to DIF. Interestingly, chirality and the substitution of fluorine with chlorine resulted in a differential reduction in virulence phenotypes. Together, this pattern of data suggests virulence-suppressing pharmacophores of DIF and structural analogues integrate halogen, hydroxyl, and carboxylic acid moiety stereochemistry. The anti-virulence effects of DIF were achieved using concentrations that are safe in humans, do not impair platelet antimicrobial functions, do not affect S. aureus growth, and do not alter the efficacy of conventional antibiotics. These results offer proof of concept for using novel anti-virulence strategies as adjuvants to antibiotic therapy to address the challenge of MRSA infection.

Diflunisal Attenuates Virulence Factor Gene Regulation and Phenotypes in Staphylococcus aureus.

Virulence factor expression is integral to pathogenicity of Staphylococcus aureus. We previously demonstrated that aspirin, through its major metabolite, salicylic acid (SAL), modulates S. aureus virulence phenotypes in vitro and in vivo. We compared salicylate metabolites and a structural analogue for their ability to modulate S. aureus virulence factor expression and phenotypes: (i) acetylsalicylic acid (ASA, aspirin); (ii) ASA metabolites, salicylic acid (SAL), gentisic acid (GTA) and salicyluric acid (SUA); or (iii) diflunisal (DIF), a SAL structural analogue. None of these compounds altered the growth rate of any strain tested. ASA and its metabolites SAL, GTA and SUA moderately impaired hemolysis and proteolysis phenotypes in multiple S. aureus strain backgrounds and their respective deletion mutants. Only DIF significantly inhibited these virulence phenotypes in all strains. The kinetic profiles of ASA, SAL or DIF on expression of hla (alpha hemolysin), sspA (V8 protease) and their regulators (sigB, sarA, agr (RNAIII)) were assessed in two prototypic strain backgrounds: SH1000 (methicillin-sensitive S. aureus; MSSA) and LAC-USA300 (methicillin-resistant S. aureus; MRSA). DIF induced sigB expression which is coincident with the significant inhibition of RNAIII expression in both strains and precedes significant reductions in hla and sspA expression. The inhibited expression of these genes within 2 h resulted in the durable suppression of hemolysis and proteolysis phenotypes. These results indicate that DIF modulates the expression of key virulence factors in S. aureus via a coordinated impact on their relevant regulons and target effector genes. This strategy may hold opportunities to develop novel antivirulence strategies to address the ongoing challenge of antibiotic-resistant S. aureus.

Staphylococcus aureus adaptive evolution: Recent insights on how immune evasion, immunometabolic subversion and host genetics impact vaccine development.

Despite meritorious attempts, a S. aureus vaccine that prevents infection or mitigates severity has not yet achieved efficacy endpoints in prospective, randomized clinical trials. This experience underscores the complexity of host-S. aureus interactions, which appear to be greater than many other bacterial pathogens against which successful vaccines have been developed. It is increasingly evident that S. aureus employs strategic countermeasures to evade or exploit human immune responses. From entering host cells to persist in stealthy intracellular reservoirs, to sensing the environmental milieu and leveraging bacterial or host metabolic products to reprogram host immune responses, S. aureus poses considerable challenges for the development of effective vaccines. The fact that this pathogen causes distinct types of infections and can undergo transient genetic, transcriptional or metabolic adaptations in vivo that do not occur in vitro compounds challenges in vaccine development. Notably, the metabolic versatility of both bacterial and host immune cells as they compete for available substrates within specific tissues inevitably impacts the variable repertoire of gene products that may or may not be vaccine antigens. In this respect, S. aureus has chameleon phenotypes that have alluded vaccine strategies thus far. Nonetheless, a number of recent studies have also revealed important new insights into pathogenesis vulnerabilities of S. aureus. A more detailed understanding of host protective immune defenses versus S. aureus adaptive immune evasion mechanisms may offer breakthroughs in the development of effective vaccines, but at present this goal remains a very high bar. Coupled with the recent advances in human genetics and epigenetics, newer vaccine technologies may enable such a goal. If so, future vaccines that protect against or mitigate the severity of S. aureus infections are likely to emerge at the intersection of precision and personalized medicine. For now, the development of S. aureus vaccines or alternative therapies that reduce mortality and morbidity must continue to be pursued.

Impacts of NaHCO3 on ß-Lactam Binding to PBP2a Protein Variants Associated with the NaHCO3-Responsive versus NaHCO3-Non-Responsive Phenotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to ß-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many ß-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed "NaHCO3-responsiveness". This phenotype features ß-lactam susceptibility of certain MRSA strains only in the presence of NaHCO3. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO3-responsive and NaHCO3-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on ß-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO3. These investigations revealed that NaHCO3 differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO3-ß-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO3. This NaHCO3-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO3-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO3-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO3-responsiveness. This investigation also provides strong evidence that the NaHCO3-responsive phenotype in MRSA may involve NaHCO3-mediated increases in both initial cell surface ß-lactam binding, as well as ultimate PBP2a binding of ß-lactams.

Mechanistic Fingerprinting Reveals Kinetic Signatures of Resistance to Daptomycin and Host Defense Peptides in Streptococcus mitis-oralis.

Streptococcus mitis-oralis (S. mitis-oralis) infections are increasingly prevalent in specific populations, including neutropenic cancer and endocarditis patients. S. mitis-oralis strains have a propensity to evolve rapid, high-level and durable resistance to daptomycin (DAP-R) in vitro and in vivo, although the mechanism(s) involved remain incompletely defined. We examined mechanisms of DAP-R versus cross-resistance to cationic host defense peptides (HDPs), using an isogenic S. mitis-oralis strain-pair: (i) DAP-susceptible (DAP-S) parental 351-WT (DAP MIC = 0.5 µg/mL), and its (ii) DAP-R variant 351-D10 (DAP MIC > 256 µg/mL). DAP binding was quantified by flow cytometry, in-parallel with temporal (1-4 h) killing by either DAP or comparative prototypic cationic HDPs (hNP-1; LL-37). Multicolor flow cytometry was used to determine kinetic cell responses associated with resistance or susceptibility to these molecules. While overall DAP binding was similar between strains, a significant subpopulation of 351-D10 cells hyper-accumulated DAP (>2-4-fold vs. 351-WT). Further, both DAP and hNP-1 induced cell membrane (CM) hyper-polarization in 351-WT, corresponding to significantly greater temporal DAP-killing (vs. 351-D10). No strain-specific differences in CM permeabilization, lipid turnover or regulated cell death were observed post-exposure to DAP, hNP-1 or LL-37. Thus, the adaptive energetics of the CM appear coupled to the outcomes of interactions of S. mitis-oralis with DAP and selected HDPs. In contrast, altered CM permeabilization, proposed as a major mechanism of action of both DAP and HDPs, did not differentiate DAP-S vs. DAP-R phenotypes in this S. mitis-oralis strain-pair.

LPA3 receptor mediates chemotaxis of immature murine dendritic cells to unsaturated lysophosphatidic acid (LPA).

Increasing evidence supports roles for lipids in the biology of immune cells. In particular, bioactive lipids such as sphingosine-1-phosphate (S1P) bind to cognate G protein-coupled receptors (GPCRs) and modulate leukocyte trafficking and homeostasis. Lysophosphatidic acid (LPA) represents a family of bioactive lipids, which differ in the length and degree of saturation of the fatty acyl chain. LPA is structurally related to S1P and exerts cellular effects by binding to five known GPCRs (LPA(1-5)). Its function in the immune system is less clear, although it was shown to induce chemotaxis of human dendritic cells (DCs) and activated T cells. In this study, we show that LPA can induce chemotaxis of immature but not mature mouse DCs and that only unsaturated and not saturated LPA species are efficient chemoattractants. However, both LPA species do not alter DC maturation or chemotaxis to other chemokines. The loss of DC migration capability correlated with the down-regulation of expression of the receptors LPA(3) and LPA(5), and expression of LPA(1), LPA(2), and LPA(4) did not change. A LPA(3) antagonist reduced immature DC migration to LPA by 70%, suggesting that LPA(3) mediates immature DC chemotaxis to unsaturated species of LPA. Furthermore, isolated, immature DCs from mice lacking LPA(3) exhibited a 50% reduction in migration to LPA. In summary, our results indicate that immature mouse DCs migrate preferentially in response to unsaturated LPA and that LPA(3) is important in this response.