Neutralizing Staphylococcus aureus Virulence with AZD6389, a Three mAb Combination, Accelerates Closure of a Diabetic Polymicrobial Wound.

Nonhealing diabetic foot ulcers (DFU), a major complication of diabetes, are associated with high morbidity and mortality despite current standard of care. Since Staphylococcus aureus is the most common pathogen isolated from nonhealing and infected DFU, we hypothesized that S. aureus virulence factors would damage tissue, promote immune evasion and alter the microbiome, leading to bacterial persistence and delayed wound healing. In a diabetic mouse polymicrobial wound model with S. aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes, we report a rapid bacterial proliferation, prolonged pro-inflammatory response and large necrotic lesions unclosed for up to 40 days. Treatment with AZD6389, a three-monoclonal antibody combination targeting S. aureus alpha toxin, 4 secreted leukotoxins, and fibrinogen binding cell-surface adhesin clumping factor A resulted in full skin re-epithelization 21 days after inoculation. By neutralizing multiple virulence factors, AZD6389 effectively blocked bacterial agglutination and S. aureus-mediated cell killing, abrogated S. aureus-mediated immune evasion and targeted the bacteria for opsonophagocytic killing. Neutralizing S. aureus virulence not only facilitated S. aureus clearance in lesions, but also reduced S. pyogenes and P. aeruginosa numbers, damaging inflammatory mediators and markers for neutrophil extracellular trap formation 14 days post initiation. Collectively, our data suggest that AZD6389 holds promise as an immunotherapeutic approach against DFU complications. IMPORTANCE Diabetic foot ulcers (DFU) represent a major complication of diabetes and are associated with poor quality of life and increased morbidity and mortality despite standard of care. They have a complex pathogenesis starting with superficial skin lesions, which often progress to deeper tissue structures up to the bone and ultimately require limb amputation. The skin microbiome of diabetic patients has emerged as having an impact on DFU occurrence and chronicity. DFU are mostly polymicrobial, and the Gram-positive bacterium Staphylococcus aureus detected in more than 95% of cases. S. aureus possess a collection of virulence factors which participate in disease progression and may facilitate growth of other pathogens. Here we show in a diabetic mouse wound model that targeting some specific S. aureus virulence factors with a multimechanistic antibody combination accelerated wound closure and promoted full skin re-epithelization. This work opens promising new avenues for the treatment of DFU.

Antibacterial Monoclonal Antibodies Do Not Disrupt the Intestinal Microbiome or Its Function.

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host's microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.

Staphylococcus aureus drives expansion of low-density neutrophils in diabetic mice.

Diabetic individuals are at considerable risk for invasive infection by Staphylococcus aureus, however, the mechanisms underlying this enhanced susceptibility to infection are unclear. We observed increased mortality following i.v. S. aureus infection in diabetic mice compared with nondiabetic controls, correlating with increased numbers of low-density neutrophils (LDNs) and neutrophil extracellular traps (NETs). LDNs have been implicated in the inflammatory pathology of diseases such as lupus, given their release of large amounts of NETs. Our goal was to describe what drives LDN increases during S. aureus infection in the diabetic host and mechanisms that promote increased NET production by LDNs. LDN development is dependent on TGF-ß, which we found to be more activated in the diabetic host. Neutralization of TGF-ß, or the TGF-ß-activating integrin αvß8, reduced LDN numbers and improved survival during S. aureus infection. Targeting S. aureus directly with MEDI4893*, an α toxin-neutralizing monoclonal antibody, blocked TGF-ß activation, reduced LDNs and NETs, and significantly improved survival. A comparison of gene and protein expression in high-density neutrophils and LDNs identified increased GPCRs and elevated phosphatase and tensin homolog (PTEN) in the LDN subset. Inhibition of PTEN improved the survival of infected diabetic mice. Our data identify a population of neutrophils in infected diabetic mice that correlated with decreased survival and increased NET production and describe 3 therapeutic targets, a bacterial target and 2 host proteins, that prevented NET production and improved survival.

The Neutrophilic Response to Pseudomonas Damages the Airway Barrier, Promoting Infection by Klebsiella pneumoniae.

Pseudomonas aeruginosa and Klebsiella pneumoniae are two common gram-negative pathogens that are associated with bacterial pneumonia and can often be isolated from the same patient. We used a mixed-pathogen pneumonia infection model in which mice were infected with sublethal concentrations of P. aeruginosa and K. pneumoniae, resulting in significant lethality, outgrowth of both bacteria in the lung, and systemic dissemination of K. pneumoniae. Inflammation, induced by P. aeruginosa activation of Toll-like receptor 5, results in prolonged neutrophil recruitment to the lung and increased levels of neutrophil elastase in the airway, resulting in lung damage and epithelial barrier dysfunction. Live P. aeruginosa was not required to potentiate K. pneumoniae infection, and flagellin alone was sufficient to induce lethality when delivered along with Klebsiella. Prophylaxis with an anti-Toll-like receptor 5 antibody or Sivelestat, a neutrophil elastase inhibitor, reduced neutrophil influx, inflammation, and mortality. Furthermore, pathogen-specific monoclonal antibodies targeting P. aeruginosa or K. pneumoniae prevented the outgrowth of both bacteria and reduced host inflammation and lethality. These findings suggest that coinfection with P. aeruginosa may enable the outgrowth and dissemination of K. pneumoniae, and that a pathogen- or host-specific prophylactic approach targeting P. aeruginosa may prevent or limit the severity of such infections by reducing neutrophil-induced lung damage.

S. aureus Evades Macrophage Killing through NLRP3-Dependent Effects on Mitochondrial Trafficking.

Clinical severity of Staphylococcus aureus respiratory infection correlates with alpha toxin (AT) expression. AT activates the NLRP3 inflammasome; deletion of Nlrp3, or AT neutralization, protects mice from lethal S. aureus pneumonia. We tested the hypothesis that this protection is not due to a reduction in inflammasome-dependent cytokines (IL-1ß/IL-18) but increased bactericidal function of macrophages. In vivo, neutralization of AT or NLRP3 improved bacterial clearance and survival, while blocking IL-1ß/IL-18 did not. Primary human monocytes were used in vitro to determine the mechanism through which NLRP3 alters bacterial killing. In cells treated with small interfering RNA (siRNA) targeting NLRP3 or infected with AT-null S. aureus, mitochondria co-localize with bacterial-containing phagosomes. Mitochondrial engagement activates caspase-1, a process dependent on complex II of the electron transport chain, near the phagosome, promoting its acidification. These data demonstrate a mechanism utilized by S. aureus to sequester itself from antimicrobial processes within the cell.

S. aureus blocks efferocytosis of neutrophils by macrophages through the activity of its virulence factor alpha toxin.

Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by "friendly fire", a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection.

Staphylococcus aureus α toxin potentiates opportunistic bacterial lung infections.

Broad-spectrum antibiotic use may adversely affect a patient's beneficial microbiome and fuel cross-species spread of drug resistance. Although alternative pathogen-specific approaches are rationally justified, a major concern for this precision medicine strategy is that co-colonizing or co-infecting opportunistic bacteria may still cause serious disease. In a mixed-pathogen lung infection model, we find that the Staphylococcus aureus virulence factor α toxin potentiates Gram-negative bacterial proliferation, systemic spread, and lethality by preventing acidification of bacteria-containing macrophage phagosomes, thereby reducing effective killing of both S. aureus and Gram-negative bacteria. Prophylaxis or early treatment with a single α toxin neutralizing monoclonal antibody prevented proliferation of co-infecting Gram-negative pathogens and lethality while also promoting S. aureus clearance. These studies suggest that some pathogen-specific, antibody-based approaches may also work to reduce infection risk in patients colonized or co-infected with S. aureus and disparate drug-resistant Gram-negative bacterial opportunists.

Anti-alpha-toxin monoclonal antibody and antibiotic combination therapy improves disease outcome and accelerates healing in a Staphylococcus aureus dermonecrosis model.

Alpha-toxin (AT) is a major virulence determinant in Staphylococcus aureus skin and soft tissue infection models. We previously demonstrated that prophylactic administration of 2A3, an AT-neutralizing monoclonal antibody (MAb), prevents S. aureus disease in a mouse dermonecrosis model by neutralizing AT-mediated tissue necrosis and immune evasion. In the present study, MEDI4893*, an affinity-optimized version of 2A3, was characterized for therapeutic activity in the dermonecrosis model as a single agent and in combination with two frontline antibiotics, vancomycin and linezolid. MEDI4893* postinfection therapy was found to exhibit a therapeutic treatment window similar to that for linezolid but longer than that for vancomycin. Additionally, when combined with either vancomycin or linezolid, MEDI4893* resulted in reduced tissue damage, increased neutrophil and macrophage infiltration and abscess formation, and accelerated healing relative to those with the antibiotic monotherapies. These data suggest that AT neutralization with a potent MAb holds promise for both prophylaxis and adjunctive therapy with antibiotics and may be a valuable addition to currently available options for the treatment of S. aureus skin and soft tissue infections.

Transmission dynamics of two strains of Schistosoma mansoni utilizing novel intermediate and definitive hosts.

The intimate host-parasite relationship mandates adaptation to the genetic and phenotypic variability of their counterparts. Here, inbred and outcrossed strains of Schistosoma mansoni were challenged with "local" and "novel" intermediate and definitive hosts to examine effects of genetic variability and novelty on infection success and dynamics. Genetically distinct lines of Biomphalaria glabrata intermediate hosts exposed to inbred and outcrossed S. mansoni larvae were assessed for differences in both snail and parasite life-history parameters. Cercariae from each parasite-snail treatment were used to infect "local" and "novel" Mus musculus definitive hosts to assess parasite infectivity and fitness. Outcrossed parasites significantly reduced snail growth, were more productive, and induced greater host mortality than inbred parasites. Mouse strain did not influence parasite infectivity or reproduction, but parasite and snail host genetic background did, affecting both sex-specific infectivity and parasite productivity. Overall, genetic background of S. mansoni and its intermediate snail host altered life history traits and transmission dynamics of the parasite throughout its life cycle.