Coordination of cohabiting phage elements supports bacteria-phage cooperation.

Bacterial pathogens often carry multiple prophages and other phage-derived elements within their genome, some of which can produce viral particles in response to stress. Listeria monocytogenes 10403S harbors two phage elements in its chromosome, both of which can trigger bacterial lysis under stress: an active prophage (ϕ10403S) that promotes the virulence of its host and can produce infective virions, and a locus encoding phage tail-like bacteriocins. Here, we show that the two phage elements are co-regulated, with the bacteriocin locus controlling the induction of the prophage and thus its activity as a virulence-associated molecular switch. More specifically, a metalloprotease encoded in the bacteriocin locus is upregulated in response to stress and acts as an anti-repressor for CI-like repressors encoded in each phage element. Our results provide molecular insight into the phenomenon of polylysogeny and its intricate adaptation to complex environments.

Phage Lysins for Fighting Bacterial Respiratory Infections: A New Generation of Antimicrobials.

Lower respiratory tract infections and tuberculosis are responsible for the death of about 4.5 million people each year and are the main causes of mortality in children under 5 years of age. Streptococcus pneumoniae is the most common bacterial pathogen associated with severe pneumonia, although other Gram-positive and Gram-negative bacteria are involved in respiratory infections as well. The ability of these pathogens to persist and produce infection under the appropriate conditions is also associated with their capacity to form biofilms in the respiratory mucous membranes. Adding to the difficulty of treating biofilm-forming bacteria with antibiotics, many of these strains are becoming multidrug resistant, and thus the alternative therapeutics available for combating this kind of infections are rapidly depleting. Given these concerns, it is urgent to consider other unconventional strategies and, in this regard, phage lysins represent an attractive resource to circumvent some of the current issues in infection treatment. When added exogenously, lysins break specific bonds of the peptidoglycan and have potent bactericidal effects against susceptible bacteria. These enzymes possess interesting features, including that they do not trigger an adverse immune response and raise of resistance is very unlikely. Although Gram-negative bacteria had been considered refractory to these compounds, strategies to overcome this drawback have been developed recently. In this review we describe the most relevant in vitro and in vivo results obtained to date with lysins against bacterial respiratory pathogens.

Streptococcus pyogenes Endopeptidase O Contributes to Evasion from Complement-mediated Bacteriolysis via Binding to Human Complement Factor C1q.

Streptococcus pyogenes secretes various virulence factors for evasion from complement-mediated bacteriolysis. However, full understanding of the molecules possessed by this organism that interact with complement C1q, an initiator of the classical complement pathway, remains elusive. In this study, we identified an endopeptidase of S. pyogenes, PepO, as an interacting molecule, and investigated its effects on complement immunity and pathogenesis. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis findings revealed that S. pyogenes recombinant PepO bound to human C1q in a concentration-dependent manner under physiological conditions. Sites of inflammation are known to have decreased pH levels, thus the effects of PepO on bacterial evasion from complement immunity was analyzed in a low pH condition. Notably, under low pH conditions, PepO exhibited a higher affinity for C1q as compared with IgG, and PepO inhibited the binding of IgG to C1q. In addition, pepO deletion rendered S. pyogenes more susceptible to the bacteriocidal activity of human serum. Also, observations of the morphological features of the pepO mutant strain (ΔpepO) showed damaged irregular surfaces as compared with the wild-type strain (WT). WT-infected tissues exhibited greater severity and lower complement activity as compared with those infected by ΔpepO in a mouse skin infection model. Furthermore, WT infection resulted in a larger accumulation of C1q than that with ΔpepO. Our results suggest that interaction of S. pyogenes PepO with C1q interferes with the complement pathway, which enables S. pyogenes to evade complement-mediated bacteriolysis under acidic conditions, such as seen in inflammatory sites.

Immunology of Yersinia pestis Infection.

As a pathogen of plague, Yersinia pestis caused three massive pandemics in history that killed hundreds of millions of people. Yersinia pestis is highly invasive, causing severe septicemia which, if untreated, is usually fatal to its host. To survive in the host and maintain a persistent infection, Yersinia pestis uses several stratagems to evade the innate and the adaptive immune responses. For example, infections with this organism are biphasic, involving an initial "noninflammatory" phase where bacterial replication occurs initially with little inflammation and following by extensive phagocyte influx, inflammatory cytokine production, and considerable tissue destruction, which is called "proinflammatory" phase. In contrast, the host also utilizes its immune system to eliminate the invading bacteria. Neutrophil and macrophage are the first defense against Yersinia pestis invading through phagocytosis and killing. Other innate immune cells also play different roles, such as dendritic cells which help to generate more T helper cells. After several days post infection, the adaptive immune response begins to provide organism-specific protection and has a long-lasting immunological memory. Thus, with the cooperation and collaboration of innate and acquired immunity, the bacterium may be eliminated from the host. The research of Yersinia pestis and host immune systems provides an important topic to understand pathogen-host interaction and consequently develop effective countermeasures.

Analysis of nanomechanical properties of Borrelia burgdorferi spirochetes under the influence of lytic factors in an in vitro model using atomic force microscopy.

BACKGROUND: Atomic force microscopy (AFM) is an experimental technique which recently has been used in biology, microbiology, and medicine to investigate the topography of surfaces and in the evaluation of mechanical properties of cells. The aim of this study was to evaluate the influence of the complement system and specific anti-Borrelia antibodies in in vitro conditions on the modification of nanomechanical features of B. burgdorferi B31 cells. MATERIAL AND METHODS: In order to assess the influence of the complement system and anti-Borrelia antibodies on B. burgdorferi s.s. B31 spirochetes, the bacteria were incubated together with plasma of identified status. The samples were applied on the surface of mica disks. Young's modulus and adhesive forces were analyzed with a NanoScope V, MultiMode 8 AFM microscope (Bruker) by the PeakForce QNM technique in air using NanoScope Analysis 1.40 software (Bruker). RESULTS/CONCLUSION: The average value of flexibility of spirochetes' surface expressed by Young's modulus was 10185.32 MPa, whereas the adhesion force was 3.68 nN. AFM is a modern tool with a broad spectrum of observational and measurement abilities. Young's modulus and the adhesion force can be treated as parameters in the evaluation of intensity and changes which take place in pathogenic microorganisms under the influence of various lytic factors. The visualization of the changes in association with nanomechanical features provides a realistic portrayal of the lytic abilities of the elements of the innate and adaptive human immune system.

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

BACKGROUND: Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. METHODS AND PRINCIPAL FINDINGS: Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. CONCLUSIONS: The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Bacterial immunotherapy of gastrointestinal tumors.

BACKGROUND: Cancer immunotherapy using bacteria dates back over 150 years. The deeper understanding on how the immune system interferes with the tumor microenvironment has led to the re-emergence of bacteria or their related products in immunotherapeutic concepts. In this review, we discuss recent approaches on experimental bacteriolytic therapy, emphasizing the specific interplay between bacteria, immune cells and tumor cells to break the tumor-induced tolerance. RESULTS: Experimental research during the last decades demonstrated beneficial but also adverse influence of bacteria on tumor growth. There is a strong correlation between chronic infections and tumor incidence. However, acute bacterial infections have favourable effects on tumor growth often contributing to complete remission. Tumor regression is usually attributable to both direct tumor cell killing (via apoptosis and/or necrosis, depending on the applied bacteria) and indirect immune stimulation. This includes (I) elimination of immunosuppressive immune cells (i.e. tumor-associated macrophages, myeloid-derived suppressor, and regulatory T cells), (II) suppression of Th2-directed cytokine secretion (TGFα, IL10), (III) providing a pro-inflammatory micro-milieu (tumor infiltrating neutrophils) and (IV) supporting the influx of cytotoxic T cells into tumors. This finally forces the development of an immunological memory and may provide long-term protection against cancer. CONCLUSION: Immunotherapy using bacteria is still a double-edged sword. Experiences from the last years have substantially contributed to when bacteria and defined components thereof might be integrated into immunotherapeutic concepts. Attempts in transferring this approach into the clinics are on their way.

Method of bacterial killing differentially affects the human innate immune response to Staphylococcus epidermidis.

BACKGROUND: In vitro investigations of human innate immune responses to extracellular bacteria commonly utilise killed preparations in preference to live organisms. The effects of the bacterial preparation method on the activation of innate signalling pathways by the common opportunistic pathogen Staphylococcus epidermidis (SE) are unknown. MATERIALS AND METHODS: Mononuclear cell cytokine expression patterns induced by live (LSE), heat-killed (HKSE) and ethanol-killed SE (EKSE) were characterized at the transcriptional and translational level. Toll-like receptor (TLR)-activating capacity of the preparations was analysed using TLR-transfected human embryonic kidney cells. RESULTS: Live SE activated NF-κB, STAT1, type I interferon, and inflammasome pathways. Killed preparations engaged the NF-κB pathway, but had significantly lower capacity to activate other innate immune pathways. CONCLUSIONS: Killing of extracellular bacteria has significant qualitative and quantitative effects on key aspects of innate responses in vitro. Interpretation of in vitro data and extrapolation of findings should take into account the potential effects of bacterial preparation and should not assume that responses to killed bacteria are predictive of responses to live organisms.

Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps.

Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.

Psoriasin (S100A7) is a major Escherichia coli-cidal factor of the female genital tract.

The female urogenital tract requires an efficient defense against bacteria, potentially derived from the adjacent intestinal tract. We have thus sought to identify the factors that protect against Escherichia coli (E. coli) in the female genital tract. Vaginal fluid from healthy human donors consistently killed E. coli in vitro and vaginal epithelium strongly expressed and secreted psoriasin. Psoriasin was constitutively produced in an organotypic vaginal epithelium model, and exposure of these cells to supernatants of E. coli cultures led to an enhanced psoriasin expression. Secreted psoriasin in vaginal fluids accounted for approximately 2.5-3% of total protein. Fractionation of vaginal fluids by high performance liquid chromatography (HPLC) showed that psoriasin co-eluted with a peak of E. coli killing activity. Our data show that normal vaginal fluid contains a powerful intrinsic antimicrobial defense against E. coli and that psoriasin contributes to the innate immune response of the female genital tract.

Complement-mediated bacteriolysis after binding of specific antibodies to drug-resistant Pseudomonas aeruginosa: morphological changes observed by using a field emission scanning electron microscope.

A bactericidal mechanism mediated by human serum was investigated by a field emission scanning electron microscope and a strain of drug-resistant Pseudomonas aeruginosa. When the bacteria were treated with meropenem, a carbapenem antibiotic, spheroplasts and bulges (spheroidization) appeared after 1-3 h. When 40% serum was added to the bacteria, the bacteria agglutinated within 2 min and then lysed after 5-30 min. Immunoelectron micrographic analyses showed dispositions of complement component C9 molecules on the cell surface of lysed bacteria by the serum treatment that might suggest formation of a membrane attack complex. Immunoglobulin G (IgG) depletion from the serum diminished the lytic activity and adding human intravenous immunoglobulin (IVIG) restored it, suggesting that lysis was induced by specific IgG binding to the bacteria. IVIG may help patients with less IgG against bacteria to overcome severe infection.

Auxiliary role for D-alanylated wall teichoic acid in Toll-like receptor 2-mediated survival of Staphylococcus aureus in macrophages.

We previously reported that Staphylococcus aureus avoids killing within macrophages by exploiting the action of Toll-like receptor 2 (TLR2), which leads to the c-Jun N-terminal kinase (JNK)-mediated inhibition of superoxide production. To search for bacterial components responsible for this event, a series of S. aureus mutants, in which the synthesis of the cell wall was interrupted, were screened for the level of JNK activation in macrophages. In addition to a mutant lacking the lipoproteins that have been suggested to act as a TLR2 ligand, two mutant strains were found to activate the phosphorylation of JNK to a lesser extent than the parental strain, and this defect was recovered by acquisition of the corresponding wild-type genes. Macrophages that had phagocytosed the mutant strains produced more superoxide than those engulfing the parental strain, and the mutant bacteria were more efficiently killed in macrophages than the parent. The genes mutated, dltA and tagO, encoded proteins involved in the synthesis of D-alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, D-alanine-bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the d-alanylated wall teichoic acid of S. aureus modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.

Identification and expression analysis of the g-type and c-type lysozymes in grass carp Ctenopharyngodon idellus.

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. We identified two genes encoding g-type and c-type lysozymes from grass carp Ctenopharyngodon idellus by the RACE method. The deduced amino acids of both lysozymes possessed typical structural residues and conserved catalytic sites similar to their counterparts across the species. In contrast, there was only 8.6% similarity of amino acid sequence between these two lysozymes. Phylogenetic analyses revealed that these two genes evolved at different rate. C-type lysozyme of grass carp was diverged early in the evolutionary history. Moreover, the expression patterns of these two genes differed. The mRNA levels of both genes were increased after bacterial infection, but the up-regulation of g-type lysozyme was much stronger than that of c-type lysozyme. We also showed that the c-type and g-type recombinant lysozymes possessed different lytic activities against fish bacterial pathogens. These results confirmed that both lysozymes play important roles in the defense of grass carp against bacterial infections. The g-type lysozyme may be induced for the defense against bacterial infections, while c-type lysozyme might be the main molecule for the house-keeping defense under normal conditions. These two types of lysozymes likely use different mechanisms to regulate their expressions.

Vibrio cholerae O139 capsular polysaccharide confers complement resistance in the absence or presence of antibody yet presents a productive target for cell lysis: implications for detection of bactericidal antibodies.

Vibrio cholerae O139 variants with different surface phenotypes have been compared for their resistance to complement and also for their susceptibility to antibody-dependent, complement-mediated bacteriolysis (ACB). While both capsular polysaccharide (CPS) and lipopolysaccharide (LPS) contribute to complement resistance in the absence of antibody, the relative survival of variants expressing only one of these surface polysaccharides reveals CPS to be of much greater significance in this respect. Variants with LPS+/CPS- or LPS-/CPS+ surface phenotypes were both susceptible to ACB, showing that antibody binding to either of the O139 polysaccharides can initiate ACB. Demonstration of the lytic potential of antibodies bound to the capsule is novel and necessitates a revision of the suggested mechanism by which CPS confers partial protection of wild-type V. cholerae O139 against ACB. This finding also raised the possibility that there may be CPS-restricted epitopes which can function as substrates for ACB, which would invalidate the common practice of using unencapsulated O139 variants for estimating bactericidal responses. Since our data did not support this scenario, we evaluated several unencapsulated strains - including variants producing normal or elongated polysaccharide chains, and a hybrid O1/O139 strain - for their utility as indicators of bactericidal antibodies to V. cholerae O139. Our findings support the use of the recently-isolated CIRS134, which ensures reproducible titrations and allows assignment of high lytic titres in assays requiring only 2% complement. However low-level, cross-serogroup lytic activity was detected when CIRS134 was used to assay responses of mice injected with O1 serogroup V. cholerae, suggesting that this indicator should be used with caution when evaluating the immunogenicity of bivalent O1/O139 vaccines.

Bacterial surface protein L binds and inactivates neutrophil proteins S100A8/A9.

Finegoldia magna is an anaerobic bacterial species that is part of the normal human flora on all nonsterile body surfaces, but it is also a significant opportunistic pathogen causing a wide range of infections. Some isolates of F. magna that are more frequently associated with clinical infection express protein L, a surface protein containing multiple homologous domains (B1-B5) that bind Igs through interactions with Ig L chains. The present study shows that the N-terminal A domain of protein L binds S100A8/A9, antibacterial proteins present in large amounts in the cytoplasm of neutrophils, but also extracellularly in tissues during inflammation. As a result, protein L-expressing F. magna are protected against killing by S100A8/A9. Igs and S100A8/A9 were found to interact independently with protein L, demonstrating that this bacterial surface protein is capable of manipulating both adaptive and innate immune defense mechanisms.

Bacteriolytic activity of the alternative pathway of complement differs kinetically from the classical pathway.

The interaction between bacterial cells and activated complement components as a kinetic biological event is described. The bacteriolytic activity of complement in human and fish serum was assayed by measuring the decrease of bioluminescence of Escherichia coli transformed with lux genes. From the kinetic curves, the bacteriolytic CB(50)- and AB(50)-units were derived at any desired time point. It was observed that these values were irregular but decreased as a function of incubation time, and reached equal values during prolonged incubation, suggesting that the difference between the classical and alternative pathway activity is kinetic. From the kinetic curves, entirely new parameters could be derived: rate of the activation phase, rate of killing by the lytic phase and rate of killing by the entire pathway in undiluted serum. The rates of human and fish classical pathway were about five and two times higher than those of the alternative pathway respectively.

Immunogenicity of a novel Stx2B-Stx1B fusion protein in a mice model of Enterohemorrhagic Escherichia coli O157:H7 infection.

Shiga toxin type 1 and 2 produced by Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are responsible for hemolytic uremic syndrome, a life-threatening sequela. We constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, designated Stx2B-Stx1B (2S for short), expressed in the E. coli BL21 and harvested the purified protein by a simple anion-exchange chromatography method. The fusion protein induced high level humoral IgG in mice, subclass analysis showed IgG1 dominate the IgG increase trend, which indicated that a partial to Th2 response contributed to this humoral reactivity. High level neutralizing antibodies elicited by this fusion protein inhibited cytotoxicity of toxins and protected mice from lethal dose challenge of lysed EHEC O157:H7.

Secreted proteins of Neisseria meningitidis protect mice against infection.

We addressed the hypothesis that meningococcal secreted proteins (MSPs) can elicit protective immunity against meningococcal disease. Endotoxin-depleted MSP preparations were used to immunise a group of 15 six-week-old BALB/c mice (25microg MSPs/dose mixed with Freund's complete adjuvant) on days 0, 14 and 21. Mice were challenged 2 weeks later with 10(7) colony forming units of live Neisseria meningitidis strain MC58 (serogroup B, ET-5). Negative and positive control groups of 15 mice each were injected with adjuvant only, or a live attenuated strain of MC58, respectively. Seven out of 15 mice (47%) from the negative control group died after 72h of challenge, whereas none of test or positive control group died. Protection afforded by the anti-MSP immune response can be at least partly attributed to complement-mediated bacterial lysis, detectable in vitro using the serum of immunised mice. Murine anti-MC58 MSP sera were bactericidal against homologous and five unrelated ET-5 serogroup B strains. However, failed to kill strains from other hypervirulent clonal lineages belonging to the same or different serogroups, despite the presence of cross-reactive antibodies detectable by immunoblotting. Similar sera raised against MSPs from an isolate belonging to the ET-37 electropherotype lineage were bactericidal against all tested isolates of this lineage and, in addition, against some but not all isolates belonging to the ET-5 lineage. FACS analysis of intact bacteria treated with anti-MSPs confirmed surface-binding of antibodies.

Mannheimia haemolytica chimeric protein vaccine composed of the major surface-exposed epitope of outer membrane lipoprotein PlpE and the neutralizing epitope of leukotoxin.

Mannheimia haemolytica is commonly identified in cattle with shipping fever pneumonia. Vaccines currently available do not provide complete protection against the disease. In an effort to develop a vaccine that delivers the immunogenic regions of leukotoxin (LKT) A and the outer membrane protein (OMP) PlpE, a total of four chimeric proteins were constructed. Mice were subcutaneously immunized with 25, 50 and 75 microg quantities of each chimeric protein. The specificity of the immune response was confirmed by Western blot analysis and enzyme-linked immunosorbent assays (ELISA). Moreover, the hyperimmune sera were bactericidal to M. haemolytica in the presence of complement and neutralized LKT. While all of the chimeric proteins induced some level of immune response two, SAC87 and SAC89, were most promising. These results demonstrate that a functional immune response against M. haemolytica can be induced by vaccination with recombinant chimeric proteins created from specific immunogenic regions of the LKT and PlpE proteins.

Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity.

Fibrinogen-related proteins (FREPs) containing fibrinogen-like (FBG) domain have been shown to be involved in immune responses in both invertebrates and vertebrates, but the underlying mechanisms remain ill-defined. In this study we isolated a cDNA encoding amphioxus (Branchiostoma belcheri) FREP homolog, BbFREP. BbFREP encoded a protein of 286 amino acids, which included a C-terminal FBG domain and clustered together with human fibrinogen beta and gamma chains. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). The recombinant BbFREP expressed in Pichia pastoris was able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displayed strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP was also able to bind to both E. coli and S. aureus. In situ hybridization indicated that BbFREP was mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response.