Significance: Staphylococcus aureus (SA) is the leading cause of skin and skin structure infections (SSSI), which are the main portal of entry for life-threatening invasive infections. Treatment failures are increasingly common due to antibiotic resistance. Importantly, SA SSSI exhibit high 1-year recurrence, despite high antibody levels against staphylococcal antigens. Therefore, critical determinants of immune protection against recurrent SA SSSI are not well understood. The present study offers important insights in this area, including: ( i ) macrophages can confer protective memory transferrable to naïve hosts; ( ii ) SA SSSI can induce memory in bone marrow myeloid precursors; and ( iii ) specific memory against recurring SA SSSI is localized and distinct from disseminated infection. These findings will accelerate novel strategies to prevent and treat SA infections.

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. Significance Neuropeptides of the central nervous system regulate neurodevelopment, metabolism, emotion, and stress responses. However, despite previously established recognition of antimicrobial activity in vitro, their precise role(s) in host defense of the brain and other contexts in vivo have been poorly understood. Here, we discovered that the neuropeptide PACAP is an evolutionarily conserved antimicrobial peptide induced in the brain in response to bacterial and fungal infection. Tissue-specific PACAP induction coincided with minimal immune cell infiltration or neuroinflammation. These findings support an emerging paradigm wherein coevolution of the immune and nervous systems is linked through ancient, multifunctional peptides having neurological and immunomodulatory functions that protect the brain. We predict that PACAP is a prototypical example of a general class of endogenous, infection-inducible host-defense neuropeptides and serves as a model template to design next-generation antiinfectives with neurohomeostatic and immunomodulatory capabilities.

Significance: The emergence of multidrug-resistant pathogens is outpacing development of new antibiotics, which remains a slow and empirical process. Although machine-learning–based metrics exist, no principle-based formulae have been applied to define α-helical host defense peptides (αHDPs). Here, knowledge-based annotation of bioinformatic databases was validated with artificial intelligence methods to converge on a common structural framework for eukaryotic αHDPs. This multidimensional structural formula successfully predicted antimicrobial activity in α-helical peptides and domains embedded in larger proteins that had no previously known antimicrobial activity. The α-core signature provides a foundational definition for evolutionary diversity among αHDPs, sheds light on host defense immunology, and may accelerate the process of antiinfective discovery and development.

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.

The role of the host in development of persistent methicillin-resistantStaphylococcus aureus(MRSA) bacteremia is not well understood. A cohort of prospectively enrolled patients with persistent methicillin-resistantS. aureusbacteremia (PB) and resolving methicillin-resistantS. aureusbacteremia (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 theDNMT3Aintronic 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 withoutDNMT3Amutation (A/C: 9.7038 pg/mL vs. A/A: 52.9898 pg/mL;P= 0.0042). Expression ofDNMT3Awas significantly suppressed in patients withS. aureusbacteremia and inS. aureus-challenged primary human macrophages. Small interfering RNA (siRNA) silencing ofDNMT3Aexpression in human macrophages caused increased IL-10 response uponS. aureusstimulation. Treating macrophages with methylation inhibitor 5-Aza-2′-deoxycytidine resulted in increased levels of IL-10 when challenged withS. aureus. In the murine sepsis model, methylation inhibition increased susceptibility toS. aureus. These findings indicate that g.25498283A > C genotype withinDNMT3Acontributes 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.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to β-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many β-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 β-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 β-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-β-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 β-lactam binding, as well as ultimate PBP2a binding of β-lactams.