Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Use of Metagenomic Next-Generation Sequencing to Identify Pathogens in Pediatric Osteoarticular Infections.

BACKGROUND: Osteoarticular infections (OAIs) are frequently encountered in children. Treatment may be guided by isolation of a pathogen; however, operative cultures are often negative. Metagenomic next-generation sequencing (mNGS) allows for broad and sensitive pathogen detection that is culture-independent. We sought to evaluate the diagnostic utility of mNGS in comparison to culture and usual care testing to detect pathogens in acute osteomyelitis and/or septic arthritis in children. METHODS: This was a single-site study to evaluate the use of mNGS in comparison to culture to detect pathogens in acute pediatric osteomyelitis and/or septic arthritis. Subjects admitted to a tertiary children's hospital with suspected OAI were eligible for enrollment. We excluded subjects with bone or joint surgery within 30 days of admission or with chronic osteomyelitis. Operative samples were obtained at the surgeon's discretion per standard care (fluid or tissue) and based on imaging and operative findings. We compared mNGS to culture and usual care testing (culture and polymerase chain reaction [PCR]) from the same site. RESULTS: We recruited 42 subjects over the enrollment period. mNGS of the operative samples identified a pathogen in 26 subjects compared to 19 subjects in whom culture identified a pathogen. In 4 subjects, mNGS identified a pathogen where combined usual care testing (culture and PCR) was negative. Positive predictive agreement and negative predictive agreement both were 93.0% for mNGS. CONCLUSIONS: In this single-site prospective study of pediatric OAI, we demonstrated the diagnostic utility of mNGS testing in comparison to culture and usual care (culture and PCR) from operative specimens.

Immune-Based Anti-Staphylococcal Therapeutic Approaches.

Widespread methicillin-resistant Staphylococcus aureus (S. aureus) infections within community and healthcare settings are responsible for accelerated development of antibiotic resistance. As the antibiotic pipeline began drying up, alternative strategies were sought for future treatment of S. aureus infections. Here, we review immune-based anti-staphylococcal strategies that, unlike conventional antibiotics, target non-essential gene products elaborated by the pathogen. These strategies stimulate narrow or broad host immune mechanisms that are critical for anti-staphylococcal defenses. Alternative approaches aim to disrupt bacterial virulence mechanisms that enhance pathogen survival or induce immunopathology. Although immune-based therapeutics are unlikely to replace antibiotics in patient treatment in the near term, they have the potential to significantly improve upon the performance of antibiotics for treatment of invasive staphylococcal diseases.

Propionibacterium acnes-induced immunopathology correlates with health and disease association.

Genomic studies revealed the existence of health- and acne-associated P. acnes strains and suggested novel approaches for broadening understanding of acne vulgaris. However, clinical association of P. acnes with disease or health has yet to be corroborated experimentally. Current animal models of acne do not closely mimic human disease and have unclear translational value. We have developed a murine model of acne by combining P. acnes inoculation with topical application of a synthetic human sebum. We showed that human sebum promoted persistence of intradermally injected P. acnes with little loss of viability after 1 week and permitted use of more physiologic inoculums. Application of acne-associated P. acnes RT4/5 strains led to development of moderate to severe skin pathology compared with application of health-associated type II P. acnes strains (RT2/6). RT4/5 P. acnes strains uniformly induced higher levels of KC (IL-8), IL-1α, IL-1ß, and IL-6 in vitro and in vivo compared with type II P. acnes strains. Overall, our data provide immunopathologic corroboration of health and disease association of clinical P. acnes strains and inform on a platform to query putative virulence factors uncovered by genomic studies.

Young bone marrow transplantation preserves learning and memory in old mice.

Restoration of cognitive function in old mice by transfer of blood or plasma from young mice has been attributed to reduced C-C motif chemokine ligand 11 (CCL11) and ß2-microglobulin, which are thought to suppress neurogenesis in the aging brain. However, the specific role of the hematopoietic system in this rejuvenation has not been defined and the importance of neurogenesis in old mice is unclear. Here we report that transplantation of young bone marrow to rejuvenate the hematopoietic system preserved cognitive function in old recipient mice, despite irradiation-induced suppression of neurogenesis, and without reducing ß2-microglobulin. Instead, young bone marrow transplantation preserved synaptic connections and reduced microglial activation in the hippocampus. Circulating CCL11 levels were lower in young bone marrow recipients, and CCL11 administration in young mice had the opposite effect, reducing synapses and increasing microglial activation. In conclusion, young blood or bone marrow may represent a future therapeutic strategy for neurodegenerative disease.

Overexpression of the C-domain of angiotensin-converting enzyme reduces melanoma growth by stimulating M1 macrophage polarization.

Angiotensin-converting enzyme (ACE) can hydrolyze many peptides and plays a central role in controlling blood pressure. Moreover, ACE overexpression in monocytes and macrophages increases resistance of mice to tumor growth. ACE is composed of two independent catalytic domains. Here, to investigate the specific role of each domain in tumor resistance, we overexpressed either WT ACE (Tg-ACE mice) or ACE lacking N- or C-domain catalytic activity (Tg-NKO and Tg-CKO mice) in the myeloid cells of mice. Tg-ACE and Tg-NKO mice exhibited strongly suppressed growth of B16-F10 melanoma because of increased ACE expression in macrophages, whereas Tg-CKO mice resisted melanoma no better than WT animals. The effect of ACE overexpression reverted to that of the WT enzyme with an ACE inhibitor but not with an angiotensin II type 1 (AT1) receptor antagonist. ACE C-domain overexpression in macrophages drove them toward a pronounced M1 phenotype upon tumor stimulation, with increased activation of NF-κB and signal transducer and activator of transcription 1 (STAT1) and decreased STAT3 and STAT6 activation. Tumor necrosis factor α (TNFα) is important for M1 activation, and TNFα blockade reverted Tg-NKO macrophages to a WT phenotype. Increased ACE C-domain expression increased the levels of reactive oxygen species (ROS) and of the transcription factor C/EBPß in macrophages, important stimuli for TNFα expression, and decreased expression of several M2 markers, including interleukin-4Rα. Natural ACE C-domain-specific substrates are not well-described, and we propose that the peptide(s) responsible for the striking ACE-mediated enhancement of myeloid function are substrates/products of the ACE C-domain.

RAS and ROS-A Story of Pseudomonas Survival.

Some pathogens block generation of reactive oxygen species to evade neutrophil killing, but how that is accomplished is poorly understood. In this issue of Cell Host & Microbe, Vareechon et al. (2017) describe ADP-ribosylation of Ras as a strategy to inhibit assembly of neutrophil NADPH oxidase.

Importance of bacillithiol in the oxidative stress response of Staphylococcus aureus.

In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.

Novel Gemini vitamin D3 analogs: large structure/function analysis and ability to induce antimicrobial peptide.

We have synthesized 39 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] analogs having two side chains attached to carbon-20 (Gemini) with various modifications and compared their anticancer activities. Five structure-function rules emerged to identify analogs with enhanced anticancer activity. One of these active analogs, BXL-01-0126, was more potent than 1,25(OH)2D3 in mediating 50% clonal inhibition of cancer cell growth. Murine studies found that BXL-01-0126 and 1,25(OH)2D3 had nearly the same potency to raise serum calcium levels. Taken together, BXL-01-0126 when compared to 1,25(OH)2D3 has greater anticancer potency, but similar toxicity causing hypercalcemia. We focused on the effect of these compounds on the stimulation of expression of human cathelicidin antimicrobial peptide (CAMP) whose gene has a vitamin D response element in its promoter. Expression of CAMP mRNA and protein increased in a dose-response fashion after exposure of acute myeloid leukemia (AML) cells to the Gemini analog, BXL-01-126, in vitro. A xenograft model of AML was developed using U937 AML cells injected into NSG-immunodeficient mice. Administration of vitamin D3 compounds to these mice resulted in substantial levels of CAMP in the systemic circulation. This suggests a unique prophylactic treatment at diagnosis or during induction chemotherapy for AML patients to provide them with protection against various microbial infections through CAMP induction.

Detection of a TLR2 agonist by hematopoietic stem and progenitor cells impacts the function of the macrophages they produce.

Several groups have shown that detection of microbial components by TLRs on hematopoietic stem and progenitor cells (HSPCs) instructs myeloid cell generation, raising interest in the possibility of targeting TLRs on HSPCs to boost myelopoiesis. However, although "TLR-derived" cells exhibit myeloid cell characteristics (phagocytosis, cytokine production, antigen presentation), it is not clear whether they are functionally equivalent to macrophages derived in the absence of TLR activation. Our in vitro and in vivo studies show that macrophages derived from mouse and human HSPC subsets (including stem cells) exposed to a TLR2 agonist prior to or during macrophage differentiation produce lower levels of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and reactive oxygen species. This is in contrast to prior exposure of differentiated macrophages to the TLR2 agonist ("tolerance"), which suppresses inflammatory cytokine production, but elevates reactive oxygen species. Soluble factors produced following exposure of HSPCs to a TLR2 agonist can also act in a paracrine manner to influence the function of macrophages derived from unexposed HSPCs. Our data demonstrate that macrophage function can be influenced by TLR signaling in the HSPCs from which they are derived, and that this may impact the clinical utility of targeting TLRs on HSPCs to boost myelopoiesis.

C/EBPε mediates nicotinamide-enhanced clearance of Staphylococcus aureus in mice.

The myeloid-specific transcription factor, CCAAT/enhancer-binding protein ε (C/EBPε) is a critical mediator of myelopoiesis. Mutation of this gene is responsible for neutrophil-specific granule deficiency in humans, a condition that confers susceptibility to Staphylococcus aureus infection. We found that C/EBPε-deficient mice are severely affected by infection with S. aureus, and C/EBPε deficiency in neutrophils contributes to the infectious phenotype. Conversely, exposure to the epigenetic modulator nicotinamide (vitamin B3) increased expression of C/EBPε in WT myeloid cells. Further, nicotinamide increased the activity of C/EBPε and select downstream antimicrobial targets, particularly in neutrophils. In a systemic murine infection model as well as in murine and human peripheral blood, nicotinamide enhanced killing of S. aureus by up to 1,000 fold but had no effect when administered to either C/EBPε-deficient mice or mice depleted of neutrophils. Nicotinamide was efficacious in both prophylactic and therapeutic settings. Our findings suggest that C/EBPε is an important target to boost killing of bacteria by the innate immune system.

The pore-forming toxin ß hemolysin/cytolysin triggers p38 MAPK-dependent IL-10 production in macrophages and inhibits innate immunity.

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns and immune-compromised adults. The pore-forming toxin (PFT) ß hemolysin/cytolysin (ßh/c) is a major virulence factor for GBS, which is generally attributed to its cytolytic functions. Here we show ßh/c has immunomodulatory properties on macrophages at sub-lytic concentrations. ßh/c-mediated activation of p38 MAPK drives expression of the anti-inflammatory and immunosuppressive cytokine IL-10, and inhibits both IL-12 and NOS2 expression in GBS-infected macrophages, which are critical factors in host defense. Isogenic mutant bacteria lacking ßh/c fail to activate p38-mediated IL-10 production in macrophages and promote increased IL-12 and NOS2 expression. Furthermore, targeted deletion of p38 in macrophages increases resistance to invasive GBS infection in mice, associated with impaired IL-10 induction and increased IL-12 production in vivo. These data suggest p38 MAPK activation by ßh/c contributes to evasion of host defense through induction of IL-10 expression and inhibition of macrophage activation, a new mechanism of action for a PFT and a novel anti-inflammatory role for p38 in the pathogenesis of invasive bacterial infection. Our studies suggest p38 MAPK may represent a new therapeutic target to blunt virulence and improve clinical outcome of invasive GBS infection.

Angiotensin-converting enzyme overexpression in mouse myelomonocytic cells augments resistance to Listeria and methicillin-resistant Staphylococcus aureus.

Gene targeting in ES cells was used to substitute control of angiotensin converting enzyme (ACE) expression from the endogenous promoter to the mouse c-fms promoter. The result is an animal model called ACE 10/10 in which ACE is overexpressed by monocytes, macrophages, and other myelomonocytic lineage cells. To study the immune response of these mice to bacterial infection, we challenged them with Listeria monocytogenes or methicillin-resistant Staphylococcus aureus (MRSA). ACE 10/10 mice have a significantly enhanced immune response to both bacteria in vivo and in vitro. For example, 5 days after Listeria infection, the spleen and liver of ACE 10/10 mice had 8.0- and 5.2-fold less bacteria than wild type mice (WT). In a model of MRSA skin infection, ACE 10/10 mice had 50-fold less bacteria than WT mice. Histologic examination showed a prominent infiltrate of ACE-positive mononuclear cells in the skin lesions from ACE 10/10. Increased bacterial resistance in ACE 10/10 is directly due to overexpression of ACE, as it is eliminated by an ACE inhibitor. Critical to increased immunity in ACE 10/10 is the overexpression of iNOS and reactive nitrogen intermediates, as inhibition of iNOS by the inhibitor 1400W eliminated all in vitro and in vivo differences in innate bacterial resistance between ACE 10/10 and WT mice. Increased resistance to MRSA was transferable by bone marrow transplantation. The overexpression of ACE and iNOS by myelomonocytic cells substantially boosts innate immunity and may represent a new means to address serious bacterial infections.

Staphylococcus aureus evades lysozyme-based peptidoglycan digestion that links phagocytosis, inflammasome activation, and IL-1beta secretion.

IL-1beta produced by phagocytes is important for protection against the mucosal pathogen Staphylococcus aureus. Processing and maturation of this cytokine requires activation of the multiprotein inflammasome complex. We observed that the bacterial cell wall component peptidoglycan (PGN) must be particulate and internalized via phagocytosis to activate NLRP3 inflammasomes and IL-1beta secretion. In the context of S. aureus infection of macrophages, we find that phagocytosis and lysozyme-based bacterial cell wall degradation are necessary to induce IL-1beta secretion. Further, an S. aureus enzyme, PGN O-acetyltransferase A, previously demonstrated to make cell wall PGN resistant to lysozyme, strongly suppresses inflammasome activation and inflammation in vitro and in vivo. These observations demonstrate that phagocytosis and lysozyme-based cell wall degradation of S. aureus are functionally coupled to inflammasome activation and IL-1beta secretion and illustrate a case whereby a bacterium specifically subverts IL-1beta secretion through chemical modification of its cell wall PGN.

Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity.

Golden color imparted by carotenoid pigments is the eponymous feature of the human pathogen Staphylococcus aureus. Here we demonstrate a role of this hallmark phenotype in virulence. Compared with the wild-type (WT) bacterium, a S. aureus mutant with disrupted carotenoid biosynthesis is more susceptible to oxidant killing, has impaired neutrophil survival, and is less pathogenic in a mouse subcutaneous abscess model. The survival advantage of WT S. aureus over the carotenoid-deficient mutant is lost upon inhibition of neutrophil oxidative burst or in human or murine nicotinamide adenine dinucleotide phosphate oxidase-deficient hosts. Conversely, heterologous expression of the S. aureus carotenoid in the nonpigmented Streptococcus pyogenes confers enhanced oxidant and neutrophil resistance and increased animal virulence. Blocking S. aureus carotenogenesis increases oxidant sensitivity and decreases whole-blood survival, suggesting a novel target for antibiotic therapy.

IKKalpha limits macrophage NF-kappaB activation and contributes to the resolution of inflammation.

Inflammation and innate immunity involve signalling pathways leading to the production of inflammatory mediators. Usually such responses are self-limiting, but aberrant resolution of inflammation results in chronic diseases. Much attention has focused on pro-inflammatory signalling but little is known about the mechanisms that resolve inflammation. The IkappaB kinase (IKK) complex contains two catalytic subunits, IKKalpha and IKKbeta, and controls the activation of NF-kappaB transcription factors, which play a pivotal role in inflammation. Ample evidence indicates that IKKbeta mediates NF-kappaB activation in response to pro-inflammatory cytokines and microbial products. IKKalpha regulates an alternative pathway important for lymphoid organogenesis, but the role of IKKalpha in inflammation is unknown. Here we describe a new role for IKKalpha in the negative regulation of macrophage activation and inflammation. IKKalpha contributes to suppression of NF-kappaB activity by accelerating both the turnover of the NF-kappaB subunits RelA and c-Rel, and their removal from pro-inflammatory gene promoters. Inactivation of IKKalpha in mice enhances inflammation and bacterial clearance. Hence, the two IKK catalytic subunits have evolved opposing but complimentary roles needed for the intricate control of inflammation and innate immunity.

Group B streptococcal beta-hemolysin/cytolysin activates neutrophil signaling pathways in brain endothelium and contributes to development of meningitis.

Meningitis occurs when blood-borne pathogens cross the blood-brain barrier (BBB) in a complex interplay between endothelial cells and microbial gene products. We sought to understand the initial response of the BBB to the human meningeal pathogen group B Streptococcus (GBS) and the organism's major virulence factors, the exopolysaccharide capsule and the beta-hemolysin/cytolysin toxin (beta-h/c). Using oligonucleotide microarrays, we found that GBS infection of human brain microvascular endothelial cells (HBMEC) induced a highly specific and coordinate set of genes including IL-8, Groalpha, Grobeta, IL-6, GM-CSF, myeloid cell leukemia sequence-1 (Mcl-1), and ICAM-1, which act to orchestrate neutrophil recruitment, activation, and enhanced survival. Most strikingly, infection with a GBS strain lacking beta-h/c resulted in a marked reduction in expression of genes involved in the immune response, while the unencapsulated strain generally induced similar or greater expression levels for the same subset of genes. Cell-free bacterial supernatants containing beta-h/c activity induced IL-8 release, identifying this toxin as a principal provocative factor for BBB activation. These findings were further substantiated in vitro and in vivo. Neutrophil migration across polar HBMEC monolayers was stimulated by GBS and its beta-h/c through a process involving IL-8 and ICAM-1. In a murine model of hematogenous meningitis, mice infected with beta-h/c mutants exhibited lower mortality and decreased brain bacterial counts compared with mice infected with the corresponding WT GBS strains.