Chronic lymphocytic leukemia immunoglobulins display bacterial reactivity that converges and diverges from auto-/poly-reactivity and IGHV mutation status.

Chronic lymphocytic leukemia (CLL) is an incurable leukemia of unknown etiology. Multiple studies suggest that the structure of the variable domains of the surface IGs on these cells, and signaling through them, play key roles in developing the disease. Hence, CLL appears to be driven by antigen-BCR interactions, and identifying the selecting antigens involved in this process is an important goal. We studied the antigen-binding characteristics of 23 CLL-derived, recombinantly-expressed IGs with 5 pathogenic bacteria, determining that CLL IGs differ in bacterial reactivity based on IGHV gene use, mutation status, and association with IGHD and IGHJ genes ("stereotypy"). Although most bacterial-reactive IGs followed the paradigm that IGHV-unmutated IGs were more auto-/poly-reactive, several did not. In addition, some CLL IGs were bacterial mono-reactive, and these displayed IGKV use biases. These findings are consistent with CLL B cells being driven into the leukemogenic process by bacterial as well as auto- antigens.

Colistin heteroresistance in Enterobacter cloacae is associated with cross-resistance to the host antimicrobial lysozyme.

Here, we describe the first identification of colistin-heteroresistant Enterobacter cloacae in the United States. Treatment of this isolate with colistin increased the frequency of the resistant subpopulation and induced cross-resistance to the host antimicrobial lysozyme. This is the first description of heteroresistance conferring cross-resistance to a host antimicrobial and suggests that clinical treatment with colistin may inadvertently select for bacteria that are resistant to components of the host innate immune system.

Limiting CDR-H3 diversity abrogates the antibody response to the bacterial polysaccharide α 1→3 dextran.

Anti-polysaccharide Ab responses in mice are often oligoclonal, and the mechanisms involved in Ag-specific clone production and selection remain poorly understood. We evaluated the relative contribution of D(H) germline content versus N nucleotide addition in a classic oligoclonal, T-independent Ab response (α 1→3 dextran [DEX]) by challenging adult TdT-sufficient (TdT(+/+)) and TdT-deficient (TdT(-/-)) gene-targeted mice, limited to the use of a single D(H) gene segment (D-limited mice), with Enterobacter cloacae. D-limited mice achieved anti-DEX-specific levels of Abs that were broadly comparable to those of wild-type (WT) BALB/c mice. Sequence analysis of the third CDR of the H chain intervals obtained by PCR amplification of V(H) domain DNA from DEX-specific plasmablasts revealed the near universal presence of an aspartic acid residue (D99) at the V-D junction, irrespective of the composition of the D(H) locus. Although WT mice were able to use germline D(H) (DQ52, DSP, or DST) gene segment sequence, TdT activity, or both to produce D99, all three D-limited mouse strains relied exclusively on N addition. Additionally, in the absence of TdT, D-limited mice failed to produce a DEX response. Coupled with previous studies demonstrating a reduced response to DEX in TdT(-/-) mice with a WT D(H) locus, we concluded that in the case of the anti-DEX repertoire, which uses a short third CDR of the H chain, the anti-DEX response relies more intensely on sequences created by postnatal N nucleotide addition than on the germline sequence of the D(H).

[Biological properties of opportunistic microorganisms--agents of acute intestinal infections].

The etiological role of conditionally pathogenic bacteria in acute intestinal infections in the Sumy region has been studied. It is established, that the strains of Klebsiella pneumoniae, Staphylococcus aureus and Enterobacter cloacae isolated from patients with acute intestinal infection were characterized by phenotypic heterogeneity. In 85.0 +/- 5.6% of the studied strains of K. pneumoniae, 30.0 +/- 7.2% of E. cloacae, 20.0 +/- 5.6% S. Aureus the authors have identified pathogenicity factors, which ensure their ability to adhesion and persistence (antiinterferon and anticomplement activity).

Absence of N addition facilitates B cell development, but impairs immune responses.

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT(-/-)) and wild-type (TdT(+/+)) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT(-/-) cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP(19)CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT(-/-) bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgM(a) and congenic TdT-sufficient CB17 IgM(b) bone marrow were placed in competition. TdT(-/-) cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.

Generation of B cell memory to the bacterial polysaccharide alpha-1,3 dextran.

B1b B cells generate a novel form of memory and provide Ab mediated-protection to persisting bacterial pathogens. To understand how B1b B cells establish memory to polysaccharide Ags, we studied an oligoclonal B cell response to alpha-1,3 dextran (DEX) expressed on Enterobacter cloacae. B cells specific for DEX enrich in the marginal zone (MZ) and B1b B cell populations. After E. cloacae immunization, MZ B cells were responsible for the generation of initial peak DEX-specific Ab titers, whereas, DEX-specific B1b B cells expanded and played an important role in boosted production of DEX-specific Ab titers upon E. cloacae rechallenge. Cell transfer experiments demonstrate that B1b B cells possess the capacity for both robust proliferation and plasma cell differentiation, thus distinguishing themselves from MZ B cells, which uniformly commit to plasma cell differentiation. These results define B1b B cells as the principal reservoir for memory to bacterial-associated polysaccharide Ags.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

Scavenger receptor B1 mediates phagocytosis and the antimicrobial peptide pathway in the endoparasitic wasp Micropilits mediator.

Scavenger receptors (SRs) are a family of pattern recognition receptors (PRRs) in the immune system. They are required for phagocytosis and act as co-receptors of Toll-like receptors to regulate immune signaling pathways in the fight against pathogens. Little is known about the function of SRs in insects. Here, we reported on a member of the SR family from the parasitic wasp Micropilits mediator (designated MmSR-B1) that is responsive to bacterial infection. The recombinant extracellular CD36 domain of MmSR-B1 produced in Escherichia coli cells is capable of binding to peptidoglycans and bacterial cells, causing agglutination of bacteria. Furthermore, we demonstrated that double-stranded RNA-mediated knockdown of MmSR-B1 impedes hemocyte phagocytosis and downregulates the expression of antimicrobial peptide (AMP) genes defensins and hymenoptaecins. Knockdown of MmSR-B1 led to increased death of the wasps when challenged by bacteria. Our study suggests that MmSR-B1 mediates phagocytosis and the production of AMPs in M. mediator wasps.

Lipids including cholesteryl linoleate and cholesteryl arachidonate contribute to the inherent antibacterial activity of human nasal fluid.

Mucosal surfaces provide first-line defense against microbial invasion through their complex secretions. The antimicrobial activities of proteins in these secretions have been well delineated, but the contributions of lipids to mucosal defense have not been defined. We found that normal human nasal fluid contains all major lipid classes (in micrograms per milliliter), as well as lipoproteins and apolipoprotein A-I. The predominant less polar lipids were myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid, cholesterol, and cholesteryl palmitate, cholesteryl linoleate, and cholesteryl arachidonate. Normal human bronchioepithelial cell secretions exhibited a similar lipid composition. Removal of less-polar lipids significantly decreased the inherent antibacterial activity of nasal fluid against Pseudomonas aeruginosa, which was in part restored after replenishing the lipids. Furthermore, lipids extracted from nasal fluid exerted direct antibacterial activity in synergism with the antimicrobial human neutrophil peptide HNP-2 and liposomal formulations of cholesteryl linoleate and cholesteryl arachidonate were active against P. aeruginosa at physiological concentrations as found in nasal fluid and exerted inhibitory activity against other Gram-negative and Gram-positive bacteria. These data suggest that host-derived lipids contribute to mucosal defense. The emerging concept of host-derived antimicrobial lipids unveils novel roads to a better understanding of the immunology of infectious diseases.

Vaccinomics to design a novel single chimeric subunit vaccine for broad-spectrum immunological applications targeting nosocomial Enterobacteriaceae pathogens.

Healthcare associated infections (HAIs) are major cause of elevated mortality, morbidity, and high healthcare costs. Development of a vaccine targeting these pathogens could benefit in reducing HAIs count and excessive use of antibiotics. This work aimed to design a multi-epitope based prophylactic/ therapeutic vaccine directing against carbapenem resistant Enterobacter cloacae and other leading nosocomial members of Enterobacteriaceae group. Based on subtractive proteomics and immunoinformatics in-depth investigation of E. cloacae reference proteome, we prioritize four targets: outer membrane usher protein-lpfC, putative outer membrane protein A-OmpA, putative outer membrane protein-FimD, and arginine transporter fulfilling criteria of vaccine candidacy. A multi-epitope peptide vaccine construct is then formulated comprising predicted epitopes with potential to evoke both innate and adaptive immunity and B-subunit of cholera toxin as an adjuvant. The construct is modelled, loop refined, improved for stability via disulfide engineering and optimized for codon usage as per Escherichia coli expression system to ensure its maximum expression. Cross-conservation analysis carried out to evaluate broad-spectrum applicability by providing cross protection against nosocomial pathogens. A blind docking method is applied further to predict predominant binding mode of the construct with TLR4 innate immune receptor, followed by molecular dynamics simulation protocol to probe complex dynamics and exposed topology of the construct epitopes for recognition and immune processing by the host. Towards the end, binding free energies of the vaccine construct-TLR4 receptor were estimated to test docking predictions and affirm complex stability. We believe these findings to be highly useful for vaccinologists in making a highly effective vaccine for E. cloacae specifically, and other notorious Enterobacteriaceae nosocomial pathogens in general.

A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria.

The immunoglobulin A (IgA) is produced to defend mucosal surfaces from environmental organisms, but host defenses against the very heavy load of intestinal commensal microorganisms are poorly understood. The IgA against intestinal commensal bacterial antigens was analyzed; it was not simply "natural antibody" but was specifically induced and responded to antigenic changes within an established gut flora. In contrast to IgA responses against exotoxins, a significant proportion of this specific anti-commensal IgA induction was through a pathway that was independent of T cell help and of follicular lymphoid tissue organization, which may reflect an evolutionarily primitive form of specific immune defense.

[Study of effect of thermolabile enterotoxin of Enterobacter cloacae on expression of cytokine genes in mice].

AIM: To study the role of production of several cytokines by macrophages and neutrophils in experiment. MATERIALS AND METHODS: Production of cytokines--IL-2, 4, 6, 10, 12, 15 as well as interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta) by macrophages and neutrophils during experiment on mice inoculated with enterotoxigenic strain of Enterobacter cloacae was measured. Expression of cytokines was determined by reverse transcription PCR and dot-hybridization on the basis of specially synthesized nucleotide sequences. RESULTS: In macrophages of experimental animals inoculated with enterotoxigenic strain of E. cloacae increased production of TGF-beta, IFN-gamma and decreased level of TNF-alpha were revealed. Transcription of mRNAs of IL-15 and IL-6 was detected in macrophages and neutrophils of experimental animals at 24 h after inoculation. Transcription of mRNAs of IL-2, IL-4 and IL-10 was detected neither in macrophages nor in neutrophils. CONCLUSION: Thermolabile enterotoxin of E. cloacae renders negative effect on IFN-gamma, promotes enhanced expression of TGF-beta and suppresses production of IL-2, IL-4, IL- 10. Absence of expression of key cytokines suggests that effect of thermolabile enterotoxin of Enterobacter bacteria excludes it from cytokine regulation of immune response during infectious process.

[Effect of Enterobacter cloacae thermolabile enterotoxin on immune system of mice].

AIM: To study the effect of Enterobactercloacae thermolabile enterotoxin (LT-enterotoxin) on different arms of immune system of mice. MATERIALS AND METHODS: Proprietary clinical strain E. cloacae 258 was used and intraperitoneal inoculation of outbred white mice with LT-enterotoxin-producing E. cloacae comprised experimental model. Phagocytic and lysosomal activity of peritoneal macrophages as well as level of APC to sheep erythrocytes were measured, spontaneous and induced NBT tests were also performed. RESULTS: Pleiotropic immunotoxic effect of LT-enterotoxin of studied bacteria was established, which characterized by suppres- sion of antigen-presenting and antigen-processing functions of macrophages, stimulation of mitotic activity of lymphocytes, decrease of activity of hexose monophosphate shunt enzymes in peritoneal macrophages as well as abnormalities in formation of specific B-lymphocytes. CONCLUSION: Obtained data allowed to reveal separate steps in pathogenesis of infectious process caused by LT-enterotoxin-producing bacteria from Enterobacter genus.

[Influence of Enterobacter toxin on the cellular immunity].

Study of dynamics of formation of spontaneous and mitogen (phytohemagglutinin - PHA, concanavalin A - ConA)-activated blast lymphocytes showed increase of number of transforming T-lymphocytes under the influence of Enterobacter cloacae thermolabile enterotoxin. Itwas noted that PHA mainly stimulated mitosis of T-cell population, ConA - of natural killers, whereas enterotoxin stimulated mitotic activity of both cell types.

Comparative analysis of peptidoglycan recognition proteins in endoparasitoid wasp Microplitis mediator.

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune receptors that specifically recognize peptidoglycans (PGNs) on the surface of a number of pathogens. Here, we have identified and characterized six PGRPs from endoparasitoid wasp, Microplitis mediator (MmePGRPs). To understand the roles of PGRPs in parasitoid wasps, we analyzed their evolutionary relationship and orthology, expression profiles during different developmental stages, and transcriptional expression following infection with Gram-positive and -negative bacteria and a fungus. MmePGRP-S1 was significantly induced in response to pathogenic infection. This prompted us to evaluate the effects of RNA interference mediated gene specific knockdown of MmePGRP-S1. The knockdown of MmePGRP-S1 (iMmePGRP-S1) dramatically affected wasps' survival following challenge by Micrococcus luteus, indicating the involvement of this particular PGRP in immune responses against Gram-positive bacteria. This action is likely to be mediated by the Toll pathway, but the mechanism remains to be determined. MmePGRP-S1 does not play a significant role in anti-fungal immunity as indicated by the survival rate of iMmePGRP-S1 wasps. This study provides a comprehensive characterization of PGRPs in the economically important hymenopteran species M. mediator.

Emerging role of lipid droplets in Aedes aegypti immune response against bacteria and Dengue virus.

In mammals, lipid droplets (LDs) are ubiquitous organelles that modulate immune and inflammatory responses through the production of lipid mediators. In insects, it is unknown whether LDs play any role during the development of immune responses. We show that Aedes aegypti Aag2 cells - an immune responsive cell lineage - accumulates LDs when challenged with Enterobacter cloacae, Sindbis, and Dengue viruses. Microarray analysis of Aag2 challenged with E.cloacae or infected with Dengue virus revealed high transcripts levels of genes associated with lipid storage and LDs biogenesis, correlating with the increased LDs numbers in those conditions. Similarly, in mosquitoes, LDs accumulate in midgut cells in response to Serratia marcescens and Sindbis virus or when the native microbiota proliferates, following a blood meal. Also, constitutive activation of Toll and IMD pathways by knocking-down their respective negative modulators (Cactus and Caspar) increases LDs numbers in the midgut. Our results show for the first time an infection-induced LDs accumulation in response to both bacterial and viral infections in Ae. Aegypti, and we propose a role for LDs in mosquito immunity. These findings open new venues for further studies in insect immune responses associated with lipid metabolism.

The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity.

Previous mosquito studies showed that the hemocyte-mediated innate immune response against Gram- Escherichia coli is phagocytosis, but against Gram+ Micrococcus sp., is melanization. We examined the immune responses mounted by Aedes aegypti towards Gram- Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium, and Gram+ Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis. Using light microscopy, electron microscopy, and survival analysis, this investigation conclusively shows that the factors governing phagocytic vs. melanization responses are complex and independent of bacterial Gram type and pathogenicity. These data provide further evidence that hemocytes are central to the immune response against prokaryotes.

Induction of immunity by latex beads and by hemolymph transfer in Galleria mellonella.

Injection of sterile latex beads into the hemocoel of last instar larvae of Galleria mellonella provoked a strong defense reaction. Cellular defense by hemocytes was followed by enhanced antibacterial activity in hemolymph. Latex-injected insects showed increased survival rates after a challenge injection with high doses of bacteria. Factors which stimulate the production of antibacterial activity could be demonstrated soon after injection by transfer of hemolymph from preinjected to untreated larvae. A large induction capacity in donor hemolymph was accompanied by a strong decrease in the total hemocyte count of free floating hemocytes, resulting from a decrease in number of plasmatocytes and granular cells, the cell types involved in the cellular defense against the injected latex beads. The results presented support the hypothesis that during cellular defense reactions, factors are released from the hemocytes which stimulate the production of antibacterial substances.

An azurocidin-like protein is induced in Trichoplusia ni larval gut cells after bacterial challenge.

Trichoplusia ni immune genes up-regulated in response to bacterial infection have been isolated using differential display polymerase chain reaction. Here we report the cloning and characterisation of a gut-specific immune gene encoding an azurocidin-like protein. The deduced protein is 317 amino acid residues long with a hydrophobic C-terminus and a predicted 17-residue signal peptide. The mature T. ni protein shows 30% identity to human azurocidin, an antibacterial protein. Like azurocidin, the T. ni protein contains two amino acid substitutions in the active site triad normally present in serine proteases. The T. ni protein was synthesised with a six-histidine C-terminal extension using the baculovirus expression system. Sequencing of the recombinant azurocidin-like protein confirmed the predicted cleavage of the signal peptide. Northern blots show that T. ni azurocidin-like protein is expressed solely in the larval gut and that expression is up-regulated by injecting or feeding bacteria. Expression reaches its highest level at 10 h after bacteria injection.

Insights into the cross-immunity mechanism within effector families of bacteria type VI secretion system from the structure of StTae4-EcTai4 complex.

The Gram-negative bacteria type VI secretion system (T6SS) has been found to play an important role in interbacterial competition, biofilm formation and many other virulence-related processes. The bacteria harboring T6SS inject the effectors into their recipient's cytoplasm or periplasm to kill them and meanwhile, to avoid inhibiting itself, the cognate immunity proteins were produced to acts as the effector inhibitor. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity (EI) pairs. We have recently solved the structures of StTae4-Tai4 and EcTae4-Tai4 complexes from the human pathogens Salmonella typhimurium and Enterobacter cloacae, respectively. It is very interesting and important to discover whether there is cross-neutralization between St- and EcTai4 and whether their effector inhibition mechanism is conserved. Here, we determined the crystal structure of StTae4 in complex with EcTai4. The solution conformation study revealed it is a compact heterotetramer that consists of an EcTai4 homodimer binding two StTae4 molecules in solution, different from that in crystal. A remarkable shift can be observed in both the flexible winding loop of StTae4 and protruding loop of EcTai4 and disulfide bonds are formed to stabilize their overall conformations. The in vitro and in vivo interactions studies showed EcTai4 can efficiently rescue the cells from the toxicity of its cognate effectors StTae4, but can not neutralize the toxic activities of the effectors from other families. These findings provide clear structural evidence to support the previous observation of cross-immunity within T6SS families and provide a basis for understanding their important roles in polymicrobial environments.