Staphylococcus aureus Depends on Eap Proteins for Preventing Degradation of Its Phenol-Soluble Modulin Toxins by Neutrophil Serine Proteases

Neutrophil granulocytes act as a first line of defense against pathogenic staphylococci. However, Staphylococcus aureus has a remarkable capacity to survive neutrophil killing, which distinguishes it from the less-pathogenic Staphylococcus epidermidis. Both species release phenol-soluble modulin (PSM) toxins, which activate the neutrophil formyl-peptide receptor 2 (FPR2) to promote neutrophil influx and phagocytosis, and which disrupt neutrophils or their phagosomal membranes at high concentrations. We show here that the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin G and proteinase 3, which are released into the extracellular space or the phagosome upon neutrophil FPR2 stimulation, effectively degrade PSMs thereby preventing their capacity to activate and destroy neutrophils. Notably, S. aureus, but not S. epidermidis, secretes potent NSP-inhibitory proteins, Eap, EapH1, EapH2, which prevented the degradation of PSMs by NSPs. Accordingly, a S. aureus mutant lacking all three NSP inhibitory proteins was less effective in activating and destroying neutrophils and it survived less well in the presence of neutrophils than the parental strain. We show that Eap proteins promote pathology via PSM-mediated FPR2 activation since murine intraperitoneal infection with the S. aureus parental but not with the NSP inhibitors mutant strain, led to a significantly higher bacterial load in the peritoneum and kidneys of mFpr2-/- compared to wild-type mice. These data demonstrate that NSPs can very effectively detoxify some of the most potent staphylococcal toxins and that the prominent human pathogen S. aureus has developed efficient inhibitors to preserve PSM functions. Preventing PSM degradation during infection represents an important survival strategy to ensure FPR2 activation.

Isolation and characterization of human monoclonal antibodies to pneumococcal capsular polysaccharide 3

The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human PPS3 antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven human PPS3-specific monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. For two humAbs, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03), C10 had fewer VL somatic mutations, higher PPS3 affinity, more ST3 opsonophagocytic and agglutinating activity, whilst both humAbs altered ST3 gene expression in vitro. After VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. In C57Bl/6 mice, C10 and C27 reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. Our findings, associate efficacy of PPS3-specific humAbs with ST3 agglutination and opsonophagocytic activity and reveal an unexpected role for the VL in functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.

Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans.

Staphylococcus aureus peptide methionine sulfoxide reductases protect from human whole blood killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing, however, Δmsr does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, Δmsr is slower to recover following co-culture with both murine and human neutrophils than USA300 wildtype. Δmsr is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wildtype and Δmsr have similar survival phenotypes when incubated with murine whole blood. However, Δmsr is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host. IMPORTANCE Oxidative stress is a host defense strategy to control bacterial infections, and bacteria have evolved systems to counteract this innate immune defense. Here we investigate the peptide methionine sulfoxide reductase system in Staphylococcus aureus that repairs oxidized methionine residues in proteins, preventing the need to resynthesize damaged proteins de novo. Most organisms have an Msr system, and in S. aureus these enzymes are protective against HOCl killing, the major oxidant produced by neutrophils. The S. aureus Msr system does not have a significant contribution to pathogenesis in bacteremia murine infection models but does protect S. aureus in both skin and intraperitoneal infection models. Strains lacking Msr activity are killed equivalently to wildtype by murine whole blood, and Δmsr is more sensitive to killing by human whole blood than the wildtype strain. These data identify the Msr enzymes as important and potentially specific factors for S. aureus pathogenesis in the human host.

Dynamics of blood morphological indicators of Wistar line rats under parenteral BLV infection

Hematological studies of Wistar rats with intraperitoneal infection of their lymphocytes from BLV-infected cows revealed markers characteristic of the leukemic process induced by the pathogen enzootic leukemia in cattle. In 75% of experimental animals, lymphocytic leukemia and neutropenia were detected. The number of lymphocytes in the blood of rats of the experimental group was 17-36 % more than in the control group, leukocytes in average by 30 %. The animals of the experimental group showed signs of erythrocyte aplasia, hemolytic or aplastic anemia. Allergy markers were observed in individual rats. This allows us to recommend an intraperitoneal method of infecting laboratory rats with suspended lymphocytes from infected livestock for rapid and informative reproduction of experimental BLV infection.

Research of low molecular fraction of C. albicans fungus cells by the ELISA in subcutaneous administration

The purpose of this work is a study of low molecular weight fraction with C. albicans protein concentrations of 1, 2, 3, and 4 mg/ml by antibody titers in subcutaneous administration. A low molecular fraction of C. albicans proteins in concentration 1, 2, 3, 4, and 5 mg/ml has been studied in white mice. Mice have subcutaneously injected 0.2 ml of the test fraction into the upper part of the right hind paw. After 14 days, 0.2 ml of test fractions were injected repeatedly into the upper part of the left hind paw. 1 month for one group and 3 months for the second group of experimental animals after the second injection intraperitoneal infection of the animals was carried out. After 14 days, determination of the protective functions of the animal body by titer of C. albicans specific antibodies has been performed by enzyme-linked immunoassay. Studies have shown that the low molecular weight fraction of C. albicans fungi cells antigens at the double subcutaneous injection of 0.2 ml does not provide the activation of immune mechanisms. The low molecular weight fraction of C. albicans proteins at subcutaneous administration does not activate the body's defense mechanisms in preventing candidiasis.

Distribution and main biological properties of Pseudomonas Aeruginosa isolates in pig farms of Krasnodar region

Bacteria of the genus Pseudomonas aeruginosa are widespread microorganisms in pig breeding enterprises of the region, and due to virulent factors and pathogenic properties, in the etiological aspect, it poses a significant threat to the body of various sex and age groups of pigs. In the form of a monoculture, Pseudomonas aeruginosa was isolated in 14.7% of cases, in association with other microorganisms - in 85.2%, and the highest proportion of Pseudomonas aeruginosa was manifested in association with Escherichia coli (47.8%), microorganisms that cause the clinic of gastrointestinal diseases in piglets during suckling and weaning periods. A feature of the epizootic manifestation of pseudomonosis in pigs in the conditions of industrial pig breeding is enzootic outbreaks. In 79.9% of cases, growth is accompanied by the formation of a water-soluble pigment of the phenotazine series pyocyanin on selective nutrient media in isolate colonies, in 80-83 % of cases, on MPA with 5 % defibrinated blood, Pseudomonas aeruginosa colonies cause hemolysis zones. Biochemical activity of P. aeruginosa cultures showed low-expressed biochemical properties, they assimilated glucose and arabinose, galactose to the formation of acid, decomposed urea, had proteolytic properties, liquefied gelatin and peptonized milk for 72 hours, showed phosphatous activity, did not form indole and H2S, cultures producing exotoxin A, with intraperitoneal infection of laboratory animals show maximum toxigenic properties. Pseudomonas aeruginosa isolates exhibit plasma-coagulating and agesive activity. The obtained results of studying the main biological properties of P. aeruginosa isolates can be used for diagnostic studies and anti-epizootic measures in the region.