Risk assessment and quantitative measurement along with monitoring of Legionella in hospital water sources.

Legionella spp. as a causative agent of Legionnaires' disease (LD) and an opportunistic pathogen creates a public health problem. Isolation and quantification of this bacteria from clinic water sources are essential for hazard appraisal and sickness avoidance. This study aimed at risk assessment and quantitative measurement along with Legionella monitoring in educational hospital water sources in Tehran, Iran. A cross-sectional study was carried out in 1 year. The conventional culture method was used in this study to isolate Legionella from water samples. The polymerase chain reaction (PCR) technique was used to confirm the identity of the isolates and ensure that they were all Legionella. Quantitative PCR (qPCR) was used to determine the count of bacteria, and HeLa cell culture was used to determine the invasion of isolates. A total of 100 water samples were collected and inoculated on GVPC (glycine, vancomycin, polymyxin, and cycloheximide) agar; 12 (12%) and 42 (42%) cases were culture and PCR positive, respectively. Percentage of Legionella presence in PCR-positive samples by the qPCR method in <103 GU/L, in about 103 and lower than 104 GU/L, and in 104 GU/L was 40.47 (17 cases), 4.76% (two cases), and 54.76% (23 cases), respectively. Invasion analysis revealed that five and four isolates had invaded HeLa cells more than twice and equally, respectively, and the others had a lower invasion than the reference strain. The findings revealed that the spread of LD in hospitals was linked to the water system. Given the importance of nosocomial infections in the medical community, establishing a hospital water monitoring system is the most effective way to control these infections, particularly Legionella infections.

Pharmacophore-guided lead optimization: the rational design of a non-zinc coordinating, sub-micromolar inhibitor of the botulinum neurotoxin serotype a metalloprotease.

Botulinum neurotoxins, responsible for the neuroparalytic syndrome botulism, are the deadliest of known biological toxins. The work described in this study was based on a three-zone pharmacophore model for botulinum neurotoxin serotype A light chain inhibition. Specifically, the pharmacophore defined a separation between the overlaps of several different, non-zinc(II)-coordinating small molecule chemotypes, enabling the design and synthesis of a new structural hybrid possessing a Ki=600 nM (+/-100 nM).

Association of Bacillus anthracis capsule with lethal toxin during experimental infection.

Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA(63)) and the residual 20-kDa fragment (PA(20)) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA(63) was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-gamma-d-glutamic acid (gamma-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/gamma-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including gamma-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for gamma-DPGA in anthrax pathogenesis.

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin.

We present evidence that Bacillus anthracis lethal toxin (LT) suppresses rather than induces proinflammatory cytokine production in macrophages. Suppression is observed with extremely low levels of LT and involves inhibition of transcription of cytokine messenger RNA. Thus, LT may contribute to anthrax pathogenesis by suppressing the inflammatory response.

Human antibodies to bacterial superantigens and their ability to inhibit T-cell activation and lethality.

Bacterial superantigens (BSAgs) cause massive stimulation of the immune system and are associated with various pathologies and diseases. To address the role of antibodies in protection against BSAgs, we screened the sera of 29 human volunteers for antibodies to the SAgs staphylococcal enterotoxin A (SEA), SEB, SEC1, and toxic shock syndrome toxin 1 (TSST-1). Although all volunteers had detectable levels of antibodies against SEB and SEC1, many (9 out of 29 volunteers) lacked detectable antibody to SEA or had minimal titers. Antibody titers to TSST-1 were well below those to SEB and SEC1, and three volunteers lacked detectable antibody to this BSAg. In addition, pooled immunoglobulin preparations obtained from different companies had antibody titers against SEs and TSST-1. There was a good correlation between antibody titers and inhibition of superantigenic effects of these toxins. Transfer of SEB-specific antibodies, obtained from pooled sera, suppressed in vitro T-cell proliferation and totally protected mice against SEB. These data suggest that the inhibitory activity of human sera was specific to antibodies directed against the toxins. Thus, it may be possible to counteract with specific antibodies BSAg-associated pathologies caused by stimulation of the immune system.

High-affinity, protective antibodies to the binding domain of botulinum neurotoxin type A.

Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A H(C). Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A H(C) was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to <0.06 nM. Epitope mapping by real-time surface plasmon resonance showed that the antibodies bound to at least two distinct regions of the BoNT/A H(C) fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.

Protection against Staphylococcus aureus sepsis by vaccination with recombinant staphylococcal enterotoxin A devoid of superantigenicity.

Staphylococcal exotoxins are virulence determinants in Staphylococcus aureus arthritis and septicemia. To assess the utility of enterotoxins as vaccine candidates for these diseases, a genetically modified staphylococcal enterotoxin A (SEA) that lacks superantigenic properties was used. Mice immunized with recombinant (r) SEA had significantly longer survival than control immunized mice and lost significantly less weight than the controls. Transfer of SEA-specific antibodies to naive mice resulted in good protection against death in staphylococcal sepsis. In vitro proliferative responses to SEA by naive lymphocytes were almost totally abolished on incubation with serum from rSEA but not with control antigen-immunized mice. These results suggest that immunization with rSEA devoid of superantigenic properties provides good protection against S. aureus sepsis. In addition, the data indicate that the protection is at least in part mediated by SEA neutralizing antibodies.

Cross-reactive antibodies prevent the lethal effects of Staphylococcus aureus superantigens.

The exotoxins produced by Staphylococcus aureus, staphylococcal enterotoxins (SE) A-E and toxic shock syndrome toxin (TSST)-1, which are associated with serious diseases, including food poisoning and toxic shock syndrome, are termed superantigens (SAgs). To examine whether common antigenic epitopes were present and whether vaccination with 1 bacterial SAg could protect against challenge with a different SE or TSST-1, mice were vaccinated with SEA, SEB, SEC1, or TSST-1 individually or in combination. Mice injected with a single toxin developed high antibody titers against other SAgs. Marked improvement in survival was observed when immunized mice were challenged with a heterologous toxin. Mice vaccinated with a mixture of toxins were fully protected against 1 or multiple toxin challenges, indicating no interference effects of multivalent vaccinations. More importantly, higher titers were found against each SAg with the multivalent vaccination than with injection with a single SAg. Thus, immunizations with 1 SAg can induce cross-protective antibodies to heterologous SAgs, and multicomponent vaccination can enhance antibody responses against each bacterial SAg.

Identifying the principal protective antigenic determinants of type A botulinum neurotoxin.

The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (Hc; residues 861-1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-Hc were isolated and cloned. Specific binding of the mAbs to BoNT/A-Hc was demonstrated by surface plasmon resonance, with Kas in the range of 10(-10) to 10(-11) M. These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin Hc fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer Hc-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the Hc-peptides had high levels of antibodies that recognized BoNT/A-Hc. However, immunizations with only one of the Hc peptides protected when mice were challenged with BoNT/A. On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.

Development of engineered vaccines effective against structurally related bacterial superantigens.

Staphylococcal and streptococcal superantigens are exotoxins that may be linked to many human pathologies involving impaired immune functions. Despite considerable sequence divergence, bacterial superantigens share extensive secondary and tertiary structure and use similar structural strategies to bind major histocompatibility complex class II receptors. We produced by site-directed mutagenesis of the conserved receptor-binding surfaces of the superantigens staphylococcal enterotoxins A and B. These vaccines protected immunized mice and rhesus monkeys from lethal toxic shock. In addition, antibodies produced against each superantigen recognized and neutralized distantly related superantigens. This antibody cross-reactivity was additive in that mixtures of superantigens used in immunization were more effective than single-component vaccines in protecting mice from challenges with individual or mixed superantigens. We conclude that an optimal combination of these genetically attenuated superantigen vaccines may protect against all structurally related superantigens.

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens.

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.

Potentiation of inhaled staphylococcal enterotoxin B-induced toxicity by lipopolysaccharide in mice.

Nonhuman primates are the established model for evaluating toxic responses to staphylococcal enterotoxins (SEs), as they react similarly to humans. Rodents are generally considered unresponsive to SEs. Binding affinities and T-cell reactivity suggest that SE binds more efficiently to primate major histocompatability complex class II receptors than to mouse receptors. We investigated the potentiation of staphylococcal enterotoxin B (SEB) inhalation toxicity by lipopolysaccharide (LPS) in BALB/c mice. Lethality occurred only when SEB was potentiated by LPS. Neither SEB nor LPS produced lethal effects alone. Temporal responses of interleukin 1 alpha, tumor necrosis factor alpha, interleukin 2, and interferon-gamma evoked by inhaled SEB were enhanced by LPS. By 24 hr after intoxication, serum cytokines decreased to baseline levels, and consistent pulmonary perivascular leukocytic infiltrates were evident histologically. Histologic lesions induced by inhalation exposure to SEB by mice, with or without potentiation by LPS, were similar to those in the rhesus monkey. Predominant pulmonary lesions included severe, diffuse interstitial and alveolar pulmonary edema, leukocytic infiltrates, mild perivascular edema, and alveolar fibrin deposition. Although the mechanism of aerosolized SEB-induced toxicity has not been completely resolved, similarities in histologic lesions, cytokine responses, and acute dose-response suggest the LPS-potentiated mouse model may be a credible alternative to the nonhuman primate model.

Superantigen vaccines: a comparative study of genetically attenuated receptor-binding mutants of staphylococcal enterotoxin A.

Superantigens exert their pathologic effects by direct binding to major histocompatibility complex (MHC) class II molecules and T cell antigen receptors (TCR), thus circumventing the normal, antigen-specific immune response. A direct link between disease and toxin suggests an excellent opportunity for vaccine intervention. Site-directed mutants of staphylococcal enterotoxin A (SEA) that have attenuated binding to either the TCR or the MHC class II molecule were developed. Both kinds of SEA mutants induced high levels of antibody against SEA when used as vaccines, and the immunized animals were fully protected when challenged with wild type toxin. However, a residual lethality was associated with the attenuated TCR-binding mutant. These results, combined with an understanding of the molecular nature of superantigen and receptor interactions, indicate that targeting MHC class II binding by site-directed mutagenesis will produce the most effective vaccine.

Protective effects of niacinamide in staphylococcal enterotoxin-B-induced toxicity.

Staphylococcal enterotoxins (SE) interact with major histocompatibility complex (MHC) class II cell-surface receptors, eliciting signal transduction in antigen-presenting cells (APC). Subsequent toxin-class II complex interaction with specific T-cell receptors induces T-cell activation. We investigated the effect of niacinamide and interleukin (IL)-10 on SEB-induced responses. In a macrophage cell line, niacinamide (ED50--2mM) and IL-10 (ED50--7U/ml) inhibited interferon (IFN)-gamma-induced MHC class II expression in a dose-dependent manner. Also, niacinamide was a potent inhibitor of T-cell proliferation induced by SEB (ED50-- 1 mM) while IL-10 has minimal effects. In mice, the temporal responses of IL-1alpha, tumor necrosis factor (TNF)-alpha, IL-2, and IFN-gamma evoked by SEB were synergistically potentiated by lipopolysaccharide (LPS). Lethality occurred only when SEB was potentiated by LPS. Niacinamide or IL-10 improved survival of mice after lethal SEB challenge. Niacinamide reduced cytokine serum levels, although the pattern differed from that of IL-10. Niacinamide primarily reduced IL-2 and IFN-gamma, while IL-10 predominantly reduced IL-1alpha and TNF-alpha. The immunomodulatory effects of niacinamide observed on SEB-induced activation of APC and T-cells in vitro and in the LPS potentiated murine model for SEB-induced toxicity suggest it may have therapeutic value.

Bacterial superantigens in human disease: structure, function and diversity.

All bacterial superantigens use common structural strategies to bind to major histocompatibility complex class II receptors, while binding the T cell antigen receptor in different ways. Overstimulation of the immune response is responsible for the acute pathological effects, while reactivation of developmentally silenced T cells might result in autoimmune disease. Certain diseases might be controlled with superantigens or genetically attenuated vaccines.

Divergence of human and nonhuman primate lymphocyte responses to bacterial superantigens.

We compared T cell responses of human, rhesus monkey (Macaca mulatta), and chimpanzee (Pan troglodytes) to four bacterial superantigens. When lymphocytes were cultured in media supplemented with species-specific sera, chimpanzee T cells were stimulated by lower doses of staphylococcal enterotoxin (SE) A and toxic shock syndrome toxin 1 (TSST1) than were human T cells, while chimpanzee responses to SEB and SEC1 were nearly equivalent to the human response. Interestingly, rhesus lymphocytes responded to 10,000 times lower amounts of SEA, SEB, and SEC1 and to 100 times lower concentrations of TSST1 than human cells. The greater sensitivity of rhesus T cells to these toxins was not a result of differences in class II binding affinities and was only partly attributable to the presence of anti-SE and TSST1 antibodies in human serum. These results suggest that rhesus T lymphocytes are more sensitive toward these bacterial superantigens than human T cells.

Staphylococcal enterotoxins A and B share a common structural motif for binding class II major histocompatibility complex molecules.

A comparative site-directed mutagenesis study of staphylococcal enterotoxins A and B was undertaken to identify key amino-acid residues which govern interactions with major histocompatibility class II molecules. This involved generating a three-dimensional homology model for enterotoxin A in complex with the HLA-DR1 molecule, based on the reported X-ray crystal structures of enterotoxin B, both free and bound to HLA-DR1. A binding motif previously described for enterotoxin B was found to be conserved in enterotoxin A. An examination of the experimental data with the homology model clarifies how T-cell responses to enterotoxin A, and most bacterial superantigens, are likely to be mediated by variations of a structurally conserved HLA-DR alpha binding motif.

Staphylococcal enterotoxin A and toxic shock syndrome toxin compete with CD4 for human major histocompatibility complex class II binding.

We have examined the role of the CD4 molecule in primary T-lymphocyte responses to the staphylococcal enterotoxins SEA, SEB, SEC1, and the toxic shock syndrome toxin TSST-1. Proliferating cells were predominantly CD4+; however, the responses to SEA and TSST-1 were most sensitive to inhibition by the anti-CD4 antibody Leu-3a. T-lymphocyte responses to the bacterial superantigens were inhibited by site-directed mutations of residues in the DR beta membrane-proximal domain (DR beta 2) that are also known to be important for interactions with CD4. SEA and TSST-1 binding to DR was reduced by the DR beta 2 mutations and by competition with soluble recombinant CD4. We propose that bacterial superantigens sequentially, or simultaneously with CD4, stabilize complexes of T-cell antigen receptors and major histocompatibility complex class II molecules. The superantigen qualities of these toxins may be due, in part, to a molecular mimicry of CD4 and other adhesion molecules.