Small-Molecule Screen Identifies Reactive Oxygen Species as Key Regulators of Neutrophil Chemotaxis

Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase–dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction

Genes down-regulated in RNase T2 mutant cells.

1<p>ATCC17978 locus tag; adjacent loci are highlighted in grey; loci associated with pili formation and motility are bolded.</p

Motility of <i>A. baumannii</i> strains.

<p><b>Panel A.</b> Images displaying the motility properties of <i>A. baumannii</i> strains 98-37-09, 98-37-09 (pWH1266; vector), ACJ7, ACJ7 (pWH1266), and ACJ7 (pACJ02; RNase T2 complementation plasmid). <b>Panel B.</b> For each strain, the diameter across the largest portion of the resulting cell motility halo was measured in millimeters, averaged (n = 3) and plotted; the Wilcoxon rank sum test (NS: not significant, i.e. <i>p</i>≥0.05, or significant at <i>p</i><0.05) are shown above each pair of strain-specific data.</p

Bacterial strains and plasmids used in this study.

1<p>Ampicillin (Amp); Kanamycin (Kan); Tetracycline (Tet), Resistant (r).</p>2<p>American Type Culture Collection (ATCC).</p

Surface colonization properties of <i>A. baumannii</i> strains.

<p>Shown are images and corresponding quantified measures of solubilized crystal violet stained <i>A. baumannii</i> wildtype, ACJ7, and RNase T2 family complemented cells to polypropylene endotracheal tubes at 37°C (<b>Panels A</b> and <b>B</b>; n = 3/group), glass cover slips (25°C; <b>Panels C</b> and <b>D</b>; n = 3/group), and stainless steel disks (25°C; <b>Panels E</b> and <b>F</b>; and 37°C; <b>Panels G</b> and <b>H</b>; n = 5/group for these disks). Asterisks indicate statistically significant differences between wildtype, ACJ7, and complement strain adherence as determined by two-way Analysis of Variance (<i>p</i><0.05). Each data set is representative of at least two independent experiments.</p

Polystyrene colonization.

<p><b>Panel A.</b> Representative images of <i>A. baumannii</i> strains ATCC17978 and 98-37-09 colonization of 24 well polystyrene microtiter plates as visualized by crystal violet staining after 48 h incubation at 37°C. <b>Panel B.</b> Representative images of <i>A. baumannii</i> strains ATCC17978, ACJ7, and 98-37-09 colonization of polystyrene tubes after 48 h incubation.</p

Zinc Pyrithione Improves the Antibacterial Activity of Silver Sulfadiazine Ointment

ABSTRACT Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus are commonly associated with biofilm-associated wound infections that are recalcitrant to conventional antibiotics. As an initial means to identify agents that may have a greater propensity to improve clearance of wound-associated bacterial pathogens, we screened a Food and Drug Administration-approved drug library for members that display bactericidal activity toward 72-h-established P. aeruginosa biofilms using an adenylate kinase reporter assay for bacterial cell death. A total of 34 compounds displayed antibiofilm activity. Among these, zinc pyrithione was also shown to reduce levels of A. baumannii and S. aureus biofilm-associated bacteria and exhibited an additive effect in combination with silver sulfadiazine, a leading topical therapeutic for wound site infections. The improved antimicrobial activity of zinc pyrithione and silver sulfadiazine was maintained in an ointment formulation and led to improved clearance of P. aeruginosa, A. baumannii, and S. aureus in a murine model of wound infection. Taken together, these results suggest that topical zinc pyrithione and silver sulfadiazine combination formulations may mitigate wound-associated bacterial infections and disease progression. IMPORTANCE Topical antimicrobial ointments ostensibly mitigate bacterial wound disease and reliance on systemic antibiotics. Yet studies have called into question the therapeutic benefits of several traditional topical antibacterials, accentuating the need for improved next-generation antimicrobial ointments. Yet the development of such agents consisting of a new chemical entity is a time-consuming and expensive proposition. Considering that drug combinations are a mainstay therapeutic strategy for the treatment of other therapeutic indications, one alternative approach is to improve the performance of conventional antimicrobial ointments by the addition of a well-characterized and FDA-approved agent. Here we report data that indicate that the antimicrobial properties of silver sulfadiazine ointments can be significantly improved by the addition of the antifungal zinc pyrithione, suggesting that such combinations may provide an improved therapeutic option for the topical treatment of wound infections

Inositol Hexakisphosphate Kinase 1 Regulates Neutrophil Function in Innate Immunity by Inhibiting Phosphatidylinositol-(3,4,5)-Trisphosphate Signaling

Inositol phosphates are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here we demonstrate that disruption of inositol hexakisphosphate kinase 1 (InsP6K1), one of the three mammalian inositol hexakisphosphate kinases (InsP6Ks) that convert inositol hexakisphosphate (InsP6) to InsP7, conferred enhanced phosphatidylinositol-(3,4,5)-trisphosphate $(PtdIns(3,4,5)P_3)$-mediated membrane translocation of the pleckstrin homology domain of the kinase Akt and thus augmented downstream $PtdIns(3,4,5)P_3$ signaling in mouse neutrophils. Consequently, these neutrophils had greater phagocytic and bactericidal ability and amplified NADPH oxidase–mediated production of superoxide. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. In contrast, an increase in intracellular InsP7 blocked chemoattractant-elicited translocation of the pleckstrin homology domain to the membrane and substantially suppressed $PtdIns(3,4,5)P_3$-mediated cellular events in neutrophils. Our findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating $PtdIns(3,4,5)P_3$ signaling in neutrophils