The alternative sigma factor SigN of Bacillus subtilis is intrinsically toxic.

Sigma factors bind and direct the RNA polymerase core to specific promoter sequences, and alternative sigma factors direct transcription of different regulons of genes. Here, we study the pBS32 plasmid-encoded sigma factor SigN of Bacillus subtilis to determine how it contributes to DNA damage-induced cell death. We find that SigN causes cell death when expressed at high levels and does so in the absence of its regulon suggesting it is intrinsically toxic. One way toxicity was relieved was by curing the pBS32 plasmid, which eliminated a positive feedback loop that led to SigN hyper-accumulation. Another way toxicity was relieved was through mutating the chromosomally encoded transcriptional repressor protein AbrB, thereby derepressing a potent antisense transcript that antagonized SigN expression. SigN efficiently competed with the vegetative sigma factor SigA in vitro, and SigN accumulation in the absence of positive feedback reduced SigA-dependent transcription suggesting that toxicity may be due to competitive inhibition of one or more essential transcripts. Why B. subtilis encodes a toxic sigma factor is unclear but SigN may function in host-inhibition during lytic conversion, as phage lysogen genes are also encoded on pBS32. IMPORTANCE Alternative sigma factors activate entire regulons of genes to improve viability in response to environmental stimuli. The pBS32 plasmid-encoded alternative sigma factor SigN of Bacillus subtilis however, is activated by the DNA damage response and leads to cellular demise. Here we find that SigN impairs viability by hyper-accumulating and outcompeting the vegetative sigma factor for the RNA polymerase core. Why B. subtilis retains a plasmid with a deleterious alternative sigma factor is unknown.

Eosinophils mediate SIgA production triggered by TLR2 and TLR4 to control Ascaris suum infection in mice.

Human ascariasis is the most prevalent but neglected tropical disease in the world, affecting approximately 450 million people. The initial phase of Ascaris infection is marked by larval migration from the host's organs, causing mechanical injuries followed by an intense local inflammatory response, which is characterized mainly by neutrophil and eosinophil infiltration, especially in the lungs. During the pulmonary phase, the lesions induced by larval migration and excessive immune responses contribute to tissue remodeling marked by fibrosis and lung dysfunction. In this study, we investigated the relationship between SIgA levels and eosinophils. We found that TLR2 and TLR4 signaling induces eosinophils and promotes SIgA production during Ascaris suum infection. Therefore, control of parasite burden during the pulmonary phase of ascariasis involves eosinophil influx and subsequent promotion of SIgA levels. In addition, we also demonstrate that eosinophils also participate in the process of tissue remodeling after lung injury caused by larval migration, contributing to pulmonary fibrosis and dysfunction in re-infected mice. In conclusion, we postulate that eosinophils play a central role in mediating host innate and humoral immune responses by controlling parasite burden, tissue inflammation, and remodeling during Ascaris suum infection. Furthermore, we suggest that the use of probiotics can induce eosinophilia and SIgA production and contribute to controlling parasite burden and morbidity of helminthic diseases with pulmonary cycles.

Intestinal IL-17R Signaling Controls Secretory IgA and Oxidase Balance in Citrobacter rodentium Infection.

Type 17 cytokines have been strongly implicated in mucosal immunity, in part by regulating the production of antimicrobial peptides. Using a mouse model of Citrobacter rodentium infection, which causes colitis, we found that intestinal IL-17RA and IL-17RC were partially required for control of infection in the colon and IL-17 regulates the production of luminal hydrogen peroxide as well as expression of Tnsf13 Reduced Tnfsf13 expression was associated with a profound defect in generating C. rodentium-specific IgA+ Ab-secreting cells. Taken together, intestinal IL-17R signaling plays key roles in controlling invading pathogens, in part by regulating luminal hydrogen peroxide as well as regulating the generation of pathogen-specific IgA+ Ab-secreting cells.

Regulation of Staphylococcal Capsule by SarZ is SigA-Dependent.

Production of capsular polysaccharides in Staphylococcus aureus is transcriptionally regulated by a control region of the cap operon that consists of SigA- and SigB-dependent promoters. A large number of regulators have been shown to affect cap gene expression. However, regulation of capsule is only partially understood. Here we found that SarZ was another regulator that activated the cap genes through the SigA-dependent promoter. Gel electrophoresis mobility shift experiments revealed that SarZ is bound to a broad region of the cap promoter including the SigA-dependent promoter but mainly the downstream region. We demonstrated that activation of cap expression by SarZ was independent of MgrA, which also activated capsule through the SigA-dependent promoter. Our results further showed that oxidative stress with hydrogen peroxide (H2O2) treatments enhanced SarZ activation of cap expression, indicating that SarZ is able to sense oxidative stress to regulate capsule production. IMPORTANCE Expression of virulence genes in Staphylococcus aureus is affected by environmental cues and is regulated by a surprisingly large number of regulators. Much is still unknown about how virulence factors are regulated by environment cues at the molecular level. Capsule is an antiphagocytic virulence factor that is highly regulated. In this study, we found SarZ was an activator of capsule and that the regulation of capsule by SarZ was affected by oxidative stress. These results provide an example of how a virulence factor could be regulated in response to an environmental cue. As the host oxidative defense system plays an important role against S. aureus, this study contributes to a better understanding of virulence gene regulation and staphylococcal pathogenesis.

Impaired salivary SIgA antibodies elicit oral dysbiosis and subsequent induction of alveolar bone loss.

OBJECTIVE: Secreted IgA (SIgA) plays a central role in preventing bacterial and viral infections on mucosal surfaces by neutralizing toxins and viruses and inhibiting bacterial attachment to epithelial cells. However, the role of salivary SIgA antibodies (Abs) in regulating oral flora is still unknown. This study aimed to evaluate the association among oral bacteria, their metabolites and periodontitis in IgA-deficient (IgA KO) and wild-type (WT) control mice. METHODS: Microcomputed tomography (micro-CT) analysis was used to assess alveolar bone resorption as a development of periodontitis. The bacterial profiles of saliva were determined using the next-generation sequencing assays. Furthermore, the metabolites in saliva were measured and compared using CE-TOFMS. RESULTS: Salivary microbiota of IgA KO mice revealed a remarkably decreased frequency of Streptococcus, and increased percentages of Aggregatibacer, Actinobacillus, and Prevotella at the genus level when compared with those of WT. Compared to WT control mice of the same age, the level of alveolar bone loss was significantly increased in IgA KO mice, and infiltration of osteoclasts was found on the surface of the alveolar bone. The metabolome profile indicated that the metabolites of IgA KO mice had greater variability in carbon metabolic, urea cycle, and lipid pathways than WT mice. CONCLUSION: These results suggest that salivary SIgA plays an important role in regulating and maintaining normal oral microflora to prevent the development of periodontal disease.

MgrA Activates Staphylococcal Capsule via SigA-Dependent Promoter.

Staphylococcus aureus capsule polysaccharide is an important antiphagocytic virulence factor. The cap genes are regulated at the promoter element (Pcap) upstream of the cap operon. Pcap, which consists of a dominant SigB-dependent promoter and a weaker upstream SigA-dependent promoter, is activated by global regulator MgrA. How MgrA activates capsule is unclear. Here, we showed that MgrA directly bound to the Pcap region and affected the SigA-dependent promoter. Interestingly, an electrophoretic mobility shift assay showed that MgrA bound to a large region of Pcap, mainly downstream of the SigA-dependent promoter. We further showed that the ArlRS two-component system and the Agr quorum sensing system activated capsule primarily through MgrA in the early growth phases.IMPORTANCE The virulence of Staphylococcus aureus depends on the expression of various virulence factors, which is governed by a complex regulatory network. We have been using capsule as a model virulence factor to study virulence gene regulation in S. aureus MgrA is one of the regulators of capsule and has a major effect on capsule production. However, how MgrA regulates capsule genes is not understood. In this study, we were able to define the mechanism involving MgrA regulation of capsule. In addition, we also delineated the role of MgrA in capsule regulatory pathways involving the key virulence regulators Agr and Arl. This study further advances our understanding of virulence gene regulation in S. aureus, an important human pathogen.

Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction.

Although both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) are present in the gut-associated lymphoid tissues (GALT), the roles of pDCs in the gut remain largely unknown. Here we show a critical role for pDCs in T cell-independent (TI) IgA production by B cells in the GALT. When pDCs of the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) (which are representative GALT) were cultured with naive B cells to induce TI IgA class switch recombination (CSR), IgA production was substantially higher than in cocultures of these cells with cDCs. IgA production was dependent on APRIL and BAFF production by pDCs. Importantly, pDC expression of APRIL and BAFF was dependent on stromal cell-derived type I IFN signaling under steady-state conditions. Our findings provide insight into the molecular basis of pDC conditioning to induce mucosal TI IgA production, which may lead to improvements in vaccination strategies and treatment for mucosal-related disorders.

Immunological evaluation of invasive Lactobacillus plantarum co-expressing EtMIC2 and chicken interleukin-18 against Eimeria tenella.

Chicken coccidiosis is a protozoan parasitic disease that leads to considerable economic losses in the poultry industry. In this study, we used invasive Lactobacillus plantarum (L.P) expressing the FnBPA protein as a novel bacterial carrier for DNA delivery into epithelial cells to develop a live oral DNA vaccine. A fusion DNA vaccine co-expressing EtMIC2 and chicken IL-18 (chIL-18) was constructed and then delivered to the host by invasive L.P. Its efficacy against Eimeria tenella challenge was evaluated in chickens by examining the relative weight gain rate; caecal lesion score; OPG; anti-coccidial index (ACI); levels of EtMIC2 antibody, FnBPA, IL-4, IL-18, IFN-γ and SIgA; and proliferation ability and percentages of CD4+ and CD8+ splenocytes. The experimental results showed that chickens immunized with invasive L.P carrying the eukaryotic expression vector pValac-EtMIC2 (pValac-EtMIC2/pSIP409-FnBPA) had markedly improved immune protection against challenge compared with that of chickens immunized with non-invasive L.P (pValac-EtMIC2/pSIP409). However, invasive L.P co-expressing EtMIC2 with the chIL-18 vector exhibited the highest protection efficiency against E. tenella. These results indicate that invasive Lactobacillus-expressing FnBPA improved humoural and cellular immunity and enhanced resistance to E. tenella. The DNA vaccine delivered by invasive Lactobacillus provides a new concept and method for the prevention of E. tenella.

Lettuce-derived secretory IgA specifically neutralizes the Shiga toxin 1 activity.

MAIN CONCLUSION: An edible plant was tested as a host for the production of secretory monoclonal IgA against Shiga toxin 1 (Stx1). The lettuce-derived IgA completely protected Vero cells from Stx1. Secretory immunoglobulin A (SIgA) is thought to control mucosal infections and thus it may be applicable to oral passive immunotherapy. Edible plants are candidate hosts for producing oral formulations with SIgA against pathogenic agents. We previously established a recombinant IgA specific for the B subunit of Shiga toxin 1 (Stx1B) consisting of the Fab fragment of Stx1B-specific monoclonal IgG and the Fc region of IgA (hyIgA). Here, we developed transgenic lettuce (Lactuca sativa) that produces hyIgA in a secretory form (S-hyIgA). An Arabidopsis-derived light-harvesting complex II (LHCB) promoter was used for the expression of all four transgenes (hyIgA heavy, light and j chains, and secretory component). Agrobacterium-mediated transformation was carried out to introduce genes into lettuce leaf discs by means of a single vector harboring all four transgenes. Consistent with the tissue specificity of the LHCB promoter, the expression of hyIgA transgenes was observed in leaf and stem tissues, which contain chloroplasts, at the mRNA and protein levels. The leaves produced hyIgA in a more than tenfold higher yield as compared with stems. The lettuce-derived S-hyIgA was found to bind to Stx1B in a dose-dependent manner by means of ELISA. A leaf extract of the transgenic lettuce completely neutralized the cytotoxicity of Stx1 against Vero cells, which are highly susceptible to Stx1. In conclusion, we established a transgenic lettuce producing a secretory form of hyIgA that can bind bacterial toxin. The results indicate that edible practical plants containing S-hyIgA will provide a possible means for immunotherapy for food poisoning.

Effects of dietary supplementation of synbiotics on growth performance, intestinal morphology, sIgA content and antioxidant capacities of broilers.

Today, several strategies are being used to decrease the serious effects of antibiotics abuse on broilers industry and public health, among which synbiotics are one of the most promising antibiotic alternative. This study was undertaken to assess the effects of synbiotics, which composed of probiotics (Bacillus subtilis) and prebiotics (xylooligosaccharide and mannanoligosaccharide), on growth performance, intestinal morphology, sIgA content and antioxidant parameters of broilers. Four hundred and fifty one-day-old commercial Cobb48 broilers were assigned to five treatments consisting of six replicates of 15 birds each pen. Five dietary treatments include basal diets (control), basal diets plus antibiotics (4 mg/kg Xanthomycin), basal diets plus 1 g of probiotics B. subtilis product/kg of diets (4 × 108  cfu/kg), basal diets plus 150 mg/kg xylooligosaccharide (35%) and 1 g/kg mannanoligosaccharide (75%), and basal diets plus synbiotics (1 g of probiotics B. subtilis product/kg of diets (4 × 108  cfu/kg), 150 mg/kg xylooligosaccharide (35%) and 1 g/kg mannanoligosaccharide (75%). The results demonstrated that on 21 and 42 days, dietary supplementation of the synbiotics significantly increased daily weight gain (p < 0.05), feed efficiency (p < 0.05), the villus height and villus:crypt ratio in the duodenum, jejunum and ileum (p < 0.05), as well as intestinal mucosa sIgA content (p < 0.05), serum T-SOD activity (p < 0.05) and lysozyme content (p < 0.05), comparing with control group. In conclusion, synbiotics (B. subtilis and xylooligosaccharide and mannanoligosaccharide) is one of the safe and ideal dietary supplementations to increase broilers' growth performance by improving small intestinal morphology, sIgA content and antioxidant capabilities.

Enhanced differentiation of intraepithelial lymphocytes in the intestine of polymeric immunoglobulin receptor-deficient mice.

To clarify the effect of secretory IgA (sIgA) deficiency on gut homeostasis, we examined intraepithelial lymphocytes (IELs) in the small intestine (SI) of polymeric immunoglobulin receptor-deficient (pIgR(-/-) ) mice. The pIgR(-/-) mice exhibited the accumulation of CD8αß(+) T-cell receptor (TCR)-αß(+) IELs (CD8αß(+) αß-IELs) after weaning, but no increase of CD8αß(+) γδ-IELs was detected in pIgR(-/-) TCR-ß(-/-) mice compared with pIgR(+/+) TCR-ß(-/-) mice. When 5-bromo-2'-deoxyuridine (BrdU) was given for 14 days, the proportion of BrdU-labelled cells in SI-IELs was not different between pIgR(+/+) mice and pIgR(-/-) mice. However, the proportion of BrdU-labelled CD8αß(+) -IELs became higher in pIgR(-/-) mice than pIgR(+/+) mice 10 days after discontinuing BrdU-labelling. Intravenously transferred splenic T cells migrated into the intraepithelial compartments of pIgR(+/+) TCR-ß(-/-) mice and pIgR(-/-) TCR-ß(-/-) mice to a similar extent. In contrast, in the case of injection of immature bone marrow cells, CD8αß(+) αß-IELs increased much more in the SI of pIgR(-/-) TCR-ß(-/-) mice than pIgR(+/+) TCR-ß(-/-) mice 8 weeks after the transfer. αß-IELs from pIgR(-/-) mice could produce more interferon-γ and interleukin-17 than those of pIgR(+/+) mice, and intestinal permeability tended to increase in the SI of pIgR(-/-) mice with aging. Taken together, these results indicate that activated CD8αß(+) αß-IELs preferentially accumulate in pIgR(-/-) mice through the enhanced differentiation of immature haematopoietic precursor cells, which may subsequently result in the disruption of epithelial integrity.

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella.

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

[THE IMMUNOLOGIC INDICATORS IN PATIENTS WITH CARIES OF CONTACT SURFACES OF LATERAL TEETH].

The samplings of patients aged 18-45 years with caries of contact surfaces of lateral teeth (n=18) and healthy adults aged 18-20 years with intact teeth (n=18) were examined The saliva taken in rotary vial on empty stomach in amount of 3-4 mi served as assay for analysis. To identify secretory immunoglobulin A, interleukin lß, interleukin-4 and interferon y saliva was centrifuged during 15 min under 1500 rpm. The supernatant fluid was analyzed using enzymoimmunoassay (test-systems "Vector-Best", Novosibirsk). The registration of reaction was implemented using multiscan Labsystem under wavelength 450 nm. The content of secretory immunoglobulin A was expressed in mg/l, cytokines - in pg/ml. It is demonstrated that in patients with caries average level of interleukin lß was almost two times higher (p<0.05) than analogous indicator in healthy examined patients. In healthy patients average level of interferon γ significantly (more than in 10 times) exceeded upper limit of allowable standard and was higher (p<0.05) in comparison with such in patients with caries of contact surfaces. The analysis of content of secretory immunoglobulin A in saliva established that in healthy patients average values of the given indicator were higher (p<0.05) than in patients with caries of contact surfaces of lateral teeth. The lower content of secretory immunoglobulin A and interferon y against the background of increased level of interleukin lß was detected in saliva of patients with caries of contact surfaces of lateral teeth. This occurrence can be considered as factor predisposing to development of caries process.

Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis.

The DevR/DosR regulator is believed to play a key role in dormancy adaptation mechanisms of Mycobacterium tuberculosis in response to a multitude of gaseous stresses, including hypoxia, which prevails within granulomas. DevR activates transcription by binding to target promoters containing a minimum of two binding sites. The proximal site overlaps with the SigA -35 element, suggesting that DevR-SigA interaction is required for activating transcription. We evaluated the roles of 14 charged residues of DevR in transcriptional activation under hypoxic stress. Seven of the 14 alanine substitution mutants were defective in regulon activation, of which K191A, R197A, and K179A+K168A (designated K179A*) mutants were significantly or completely compromised in DNA binding. Four mutants, namely, E154A, R155A, E178A, and K208A, were activation defective in spite of binding to DNA and were classified as positive-control (pc) mutants. The SigA interaction defect of the E154A and E178A proteins was established by in vitro and in vivo assays and implies that these substitutions lead to an activation defect because they disrupt an interaction(s) with SigA. The relevance of DevR interaction to the transcriptional machinery was further established by the hypoxia survival phenotype displayed by SigA interaction-defective mutants. Our findings demonstrate the role of DevR-SigA interaction in the activation mechanism and in bacterial survival under hypoxia and establish the housekeeping sigma factor SigA as a molecular target of DevR. The interaction of DevR and RNA polymerase suggests a new and novel interceptable molecular interface for future antidormancy strategies for Mycobacterium tuberculosis.

Matrine provides a protective effect against Eimeria tenella challenge by alleviating intestinal barrier damage.

As a virulent and harmful protozoan, Eimeria tenella (E.tenella) causes harmful chicken coccidiosis, inducing high economic losses in the chicken industry. The management of the coccidial disease has been greatly hampered by drug resistance. Matrine is an active ingredient from Ku Shen (Radix Sophorae Flavescentis), a typical pesticide in chinese medicine. The aim of this study was to examine matrine's possible effectiveness in the treatment of coccidiosis and its protective function on the intestinal barrier. The anticoccidial index (ACI), the levels of anti-oxidant indexes, and secretory immunoglobulin A (sIgA) were detected. The levels of mRNA and protein expression of Occludin, ZO-1, and Claudin-1 were determined through quantitative real-time PCR (RT-qPCR) and immunohistochemistry (IHC) analysis. Matrine exhibited a moderate ACI value, and ACI values of 122.51 and 143.42 corresponded to 5 and 10 mg/kg of matrine, respectively. Compared to the infective control group, the expression of tight junction proteins significantly increased in the matrine-treatment group by RT-PCR and IHC analysis, which are essential for the mucosal immune system and the intestinal barrier. Besides, the matrine-treatment group showed a more complete intestinal structure, fewer bleeding spots, and coccidian by histopathology analysis. We also found that, matrine significantly enhanced the antioxidant ability and significantly increased the content of sIgA. Above all, matrine was considered an efficient drug against E.tenella by the anti-oxidant efficacy, and the ability to protect the composition and function of the intestinal barrier.

Secretory-IgA antibodies play an important role in the immunity to Streptococcus pneumoniae.

This study was designed to investigate whether secretory-IgA (S-IgA) Abs induced by a pneumococcal surface protein A (PspA)-based nasal vaccine are necessary for prevention of streptococcal colonization. Mice nasally immunized with PspA plus a plasmid expressing Flt3 ligand (pFL) cDNA as a mucosal adjuvant showed significantly higher levels of PspA-specific S-IgA and IgG Ab responses in both plasma and nasal washes when compared with naive mice. Although IgA(-/-) mice given nasal PspA plus pFL had significantly high levels of PspA-specific IgG Abs, high numbers of CFUs were detected in nasal washes and nasal passages. In contrast, vaccinated wild-type mice showed essentially no bacteria in the nasal cavity. Further, a nasal vaccine consisting of PspA plus pFL effectively reduced pre-existing Streptococcus pneumoniae in the nasal cavity. These results show that PspA-based vaccine-induced specific S-IgA Abs play a necessary role in the regulation of S. pneumoniae colonization in the nasal cavity.

Isolation and oral immunogenicity assessment of porcine epidemic diarrhea virus NH-TA2020 strain: One of the predominant strains circulating in China from 2017 to 2021.

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets. Maternal vaccines can effectively enhance the gut-mammary gland-secretory IgA axis to boost lactogenic immunity and passive protection of nursing piglets against PEDV challenge. From 2017 to 2021, we collected 882 diarrhea samples from 303 farms in China to investigate the epidemiology of PEDV. The result showed that about 52.15% (158/303) of the farms were positive for PEDV with an overall detection rate of 63.95% (564/882) of the samples. The S1 fragments of S gene from 104 strains were sequenced for the phylogenetic analysis. A total of 71 PEDV strains (68.27%) sequenced in this study were clustered into the predominant G2c subgroup, while the newly-defined G2d strains (9.62%) were identified in three provinces of China. The NH-TA2020 strain of G2c subgroup was isolated and cultured, and its infection to piglets caused watery diarrhea within 24 â€‹h, indicating its strong pathogenicity. Oral administration of NH-TA2020 strain to pregnant gilts stimulated high levels of IgA antibody in colostrum. The piglets fed by the gilts above were challenged with NH-TA2020 strain or CH-HeB-RY-2020 strain from G2d subgroup, and the clinical symptoms and virus shedding were significantly reduced compared to the mock group. Our findings suggest that G2c subgroup is the predominant branch circulating in China from 2017 to 2021. Oral administration of NH-TA2020 enhances maternal IgA and lactogenic immune responses, which confer protection against the homologous and emerging G2d PEDV strains challenges in neonates.

Expression, Purification, and In Silico Characterization of Mycobacterium smegmatis Alternative Sigma Factor SigB.

Sigma factor B (SigB), an alternative sigma factor (ASF), is very similar to primary sigma factor SigA (σ 70) but dispensable for growth in both Mycobacterium smegmatis (Msmeg) and Mycobacterium tuberculosis (Mtb). It is involved in general stress responses including heat, oxidative, surface, starvation stress, and macrophage infections. Despite having an extremely short half-life, SigB tends to operate downstream of at least three stress-responsive extra cytoplasmic function (ECF) sigma factors (SigH, SigE, SigL) and SigF involved in multiple signaling pathways. There is very little information available regarding the regulation of SigB sigma factor and its interacting protein partners. Hence, we cloned the SigB gene into pET28a vector and optimized its expression in three different strains of E. coli, viz., (BL21 (DE3), C41 (DE3), and CodonPlus (DE3)). We also optimized several other parameters for the expression of recombinant SigB including IPTG concentration, temperature, and time duration. We achieved the maximum expression of SigB at 25°C in the soluble fraction of the cell which was purified by affinity chromatography using Ni-NTA and further confirmed by Western blotting. Further, structural characterization demonstrates the instability of SigB in comparison to SigA that is carried out using homology modeling and structure function relationship. We have done protein-protein docking of RNA polymerase (RNAP) of Msmeg and SigB. This effort provides a platform for pulldown assay, structural, and other studies with the recombinant protein to deduce the SigB interacting proteins, which might pave the way to study its signaling networks along with its regulation.

Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans.

A functional comparison was made between a monoclonal secretory antibody generated in transgenic plants and its parent murine IgG antibody.The affinity constants of both antibodies for a Streptococcus mutans adhesion protein were similar. However the secretory antibody had a higher functional affinity due to its dimeric structure. In the human oral cavity, the secretory antibody survived for up to three days, compared with one day for the IgG antibody. The plant secretory antibody afforded specific protection in humans against oral streptococcal colonization for at least four months. We demonstrate that transgenic plants can be used to produce high affinity, monoclonal secretory antibodies that can prevent specific microbial colonization in humans. These findings could be extended to the immunotherapeutic prevention of other mucosal infections in humans and animals.

IgA Plasma Cells Are Long-Lived Residents of Gut and Bone Marrow That Express Isotype- and Tissue-Specific Gene Expression Patterns.

Antibody secreting plasma cells are made in response to a variety of pathogenic and commensal microbes. While all plasma cells express a core gene transcription program that allows them to secrete large quantities of immunoglobulin, unique transcriptional profiles are linked to plasma cells expressing different antibody isotypes. IgA expressing plasma cells are generally thought of as short-lived in mucosal tissues and they have been understudied in systemic sites like the bone marrow. We find that IgA+ plasma cells in both the small intestine lamina propria and the bone marrow are long-lived and transcriptionally related compared to IgG and IgM expressing bone marrow plasma cells. IgA+ plasma cells show signs of shared clonality between the gut and bone marrow, but they do not recirculate at a significant rate and are found within bone marrow plasma cells niches. These data suggest that systemic and mucosal IgA+ plasma cells are from a common source, but they do not migrate between tissues. However, comparison of the plasma cells from the small intestine lamina propria to the bone marrow demonstrate a tissue specific gene transcription program. Understanding how these tissue specific gene networks are regulated in plasma cells could lead to increased understanding of the induction of mucosal versus systemic antibody responses and improve vaccine design.