Roles of S100A8, S100A9 and S100A12 in infection, inflammation and immunity.

S100 proteins are small proteins that are only expressed in vertebrates. They are widely expressed in many different cell types and are involved in the regulation of calcium homeostasis, glucose metabolism, cell proliferation, apoptosis, inflammation and tumorigenesis. As members of the S100 protein subfamily of myeloid-related proteins, S100A8, S100A9 and S100A12 play a crucial role in resisting microbial infection and maintaining immune homeostasis. These proteins chelate the necessary metal nutrients of pathogens invading the host by means of 'nutritional immunity' and directly inhibit the growth of pathogens in the host. They interact with receptors on the cell surface to initiate inflammatory signal transduction, induce cytokine expression and participate in the inflammatory response and immune regulation. Furthermore, the increased content of these proteins during the pathological process makes them useful as disease markers for screening and detecting related diseases. This article summarizes the structure and function of the proteins S100A8, S100A9 and S100A12 and lays the foundation for further understanding their roles in infection, immunity and inflammation, as well as their potential applications in the prevention and treatment of infectious diseases.

Generation and Application of Monoclonal Antibodies against Porcine S100A8, S100A9, and S100A12 Proteins Using Hybridoma Technology.

S100A8, S100A9, and S100A12 proteins are important members of the S100 protein family, act primarily as congenital immunomodulators, and are closely related to the occurrence of infectious diseases. There have been few reports on the functional properties of S100A8, S100A9, and S100A12 proteins in swine, but it is certain that porcine S100A8, S100A9, and S100A12 proteins are highly expressed in diseased swine. To address the current lack of reliable and timely detection tools for these three proteins, we generated monoclonal antibodies specific to the porcine S100A8, S100A9, and S100A12 proteins using hybridoma technology. The results of serum sample testing showed that the above monoclonal antibodies specifically recognize the proteins S100A8, S100A9, and S100A12 in the serum and were able to evaluate the content change of these proteins during the infection process. This provides the basis for the use of porcine S100A8, S100A9, and S100A12 in the surveillance and diagnosis of swine diseases and laid a foundation for further understanding their roles in infection, immunity, and inflammation, as well as their potential applications in preventing or treating gastrointestinal tract or inflammatory diseases in swine.

The Love and Hate Relationship between T5SS and Other Secretion Systems in Bacteria.

Bacteria have existed on Earth for billions of years, exhibiting ubiquity and involvement in various biological activities. To ensure survival, bacteria usually release and secrete effector proteins to acquire nutrients and compete with other microorganisms for living space during long-term evolution. Consequently, bacteria have developed a range of secretion systems, which are complex macromolecular transport machines responsible for transporting proteins across the bacterial cell membranes. Among them, one particular secretion system that stands out from the rest is the type V secretion system (T5SS), known as the "autotransporter". Bacterial activities mediated by T5SS include adherence to host cells or the extracellular matrix, invasion of host cells, immune evasion and serum resistance, contact-dependent growth inhibition, cytotoxicity, intracellular flow, protease activity, autoaggregation, and biofilm formation. In a bacterial body, it is not enough to rely on T5SS alone; in most cases, T5SS cooperates with other secretion systems to carry out bacterial life activities, but regardless of how good the relationship is, there is friction between the secretion systems. T5SS and T1SS/T2SS/T3SS/T6SS all play a synergistic role in the pathogenic processes of bacteria, such as nutrient acquisition, pathogenicity enhancement, and immune modulation, but T5SS indirectly inhibits the function of T4SS. This could be considered a love-hate relationship between secretion systems. This paper uses the systematic literature review methodology to review 117 journal articles published within the period from 1995 to 2024, which are all available from the PubMed, Web of Science, and Scopus databases and aim to elucidate the link between T5SS and other secretion systems, providing clues for future prevention and control of bacterial diseases.

Bacterial and Viral Co-Infection in the Intestine: Competition Scenario and Their Effect on Host Immunity.

Bacteria and viruses are both important pathogens causing intestinal infections, and studies on their pathogenic mechanisms tend to focus on one pathogen alone. However, bacterial and viral co-infections occur frequently in clinical settings, and infection by one pathogen can affect the severity of infection by another pathogen, either directly or indirectly. The presence of synergistic or antagonistic effects of two pathogens in co-infection can affect disease progression to varying degrees. The triad of bacterial-viral-gut interactions involves multiple aspects of inflammatory and immune signaling, neuroimmunity, nutritional immunity, and the gut microbiome. In this review, we discussed the different scenarios triggered by different orders of bacterial and viral infections in the gut and summarized the possible mechanisms of synergy or antagonism involved in their co-infection. We also explored the regulatory mechanisms of bacterial-viral co-infection at the host intestinal immune interface from multiple perspectives.

Functions of the Zinc-Sensing Receptor GPR39 in Regulating Intestinal Health in Animals.

G protein-coupled receptor 39 (GPR39) is a zinc-sensing receptor (ZnR) that can sense changes in extracellular Zn2+, mediate Zn2+ signal transmission, and participate in the regulation of numerous physiological activities in living organisms. For example, GPR39 activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol3-kinase/protein kinase B (PI3K/AKT) signaling pathways upon Zn2+ stimulation, enhances the proliferation and differentiation of colonic cells, and regulates ion transport, as well as exerting other functions. In recent years, with the increased attention to animal gut health issues and the intensive research on GPR39, GPR39 has become a potential target for regulating animal intestinal health. On the one hand, GPR39 is involved in regulating ion transport in the animal intestine, mediating the Cl- efflux by activating the K+/Cl- synergistic protein transporter, and relieving diarrhea symptoms. On the other hand, GPR39 can maintain the homeostasis of the animal intestine, promoting pH restoration in colonic cells, regulating gastric acid secretion, and facilitating nutrient absorption. In addition, GPR39 can affect the expression of tight junction proteins in intestinal epithelial cells, improving the barrier function of the animal intestinal mucosa, and maintaining the integrity of the intestine. This review summarizes the structure and signaling transduction processes involving GPR39 and the effect of GPR39 on the regulation of intestinal health in animals, with the aim of further highlighting the role of GPR39 in regulating animal intestinal health and providing new directions and ideas for studying the prevention and treatment of animal intestinal diseases.

Mechanism of nitrite transporter NirC in motility, biofilm formation, and adhesion of avian pathogenic Escherichia coli.

The Escherichia coli (E. coli) nirC gene encodes a nitrite transporter, which involved in transporting toxic nitrite (NO2-) from the environment into the bacteria. Although the deletion of nirC gene could cause changes in motility, adhesion in the previous study, and the virulence involved in the specified mechanism for pathogenic E. coli remains to be known. In the present work, we aimed to evaluate the role of NirC in a serotype O2:K1:H7 avian pathogenic Escherichia coli (APEC) strain. For this purpose, we generated a NirC-deficient mutant of APEC XM strain and examined its biological characteristics. The nirC gene deletion mutant enhanced ability of motility, decreased in biofilm formation, and it markedly reduced ability to adhere mouse brain microvascular endothelial cell b.End3 cells. For understanding its mechanism, sequentially we detected and found the stress regulator rpoS and its downstream genes csrA were up-regulated in NirC-deficient mutant while diguanylate cyclase gene dgcT was down-regulated. By high-performance liquid chromatography (HPLC) experiment, we demonstrated the concentration of intracellular 3',5'-cyclic diguanosine monophosphate (c-di-GMP) significantly decrease in nirC gene deletion mutant. Taken data together, we may make a conclusion with a possible signal pathway clue, due to NirC mutation, environmental NO2- accumulation leads to nitrite stress and inactivates c-di-GMP synthesis by stimulating the stress regulator RpoS, resulting in changes of biological characteristics.

Zinc is an important inter-kingdom signal between the host and microbe.

Zinc (Zn) is an essential trace element in living organisms and plays a vital role in the regulation of both microbial virulence and host immune responses. A growing number of studies have shown that zinc deficiency or the internal Zn concentration does not meet the needs of animals and microbes, leading to an imbalance in zinc homeostasis and intracellular signalling pathway dysregulation. Competition for zinc ions (Zn2+) between microbes and the host exists in the use of Zn2+ to maintain cell structure and physiological functions. It also affects the interplay between microbial virulence factors and their specific receptors in the host. This review will focus on the role of Zn in the crosstalk between the host and microbe, especially for changes in microbial pathogenesis and nociceptive neuron-immune interactions, as it may lead to new ways to prevent or treat microbial infections.

Positive regulation of Type III secretion effectors and virulence by RyhB paralogs in Salmonella enterica serovar Enteritidis.

Small non-coding RNA RyhB is a key regulator of iron homeostasis in bacteria by sensing iron availability in the environment. Although RyhB is known to influence bacterial virulence by interacting with iron metabolism related regulators, its interaction with virulence genes, especially the Type III secretion system (T3SS), has not been reported. Here, we demonstrate that two RyhB paralogs of Salmonella enterica serovar Enteritidis upregulate Type III secretion system (T3SS) effectors, and consequently affect Salmonella invasion into intestinal epithelial cells. Specifically, we found that RyhB-1 modulate Salmonella response to stress condition of iron deficiency and hypoxia, and stress in simulated intestinal environment (SIE). Under SIE culture conditions, both RyhB-1 and RyhB-2 are drastically induced and directly upregulate the expression of T3SS effector gene sipA by interacting with its 5' untranslated region (5' UTR) via an incomplete base-pairing mechanism. In addition, the RyhB paralogs upregulate the expression of T3SS effector gene sopE. By regulating the invasion-related genes, RyhBs in turn affect the ability of S. Enteritidis to adhere to and invade into intestinal epithelial cells. Our findings provide evidence that RyhBs function as critical virulence factors by directly regulating virulence-related gene expression. Thus, inhibition of RyhBs may be a potential strategy to attenuate Salmonella.

Sef fimbria operon construction, expression, and function for direct rapid detection of Salmonella Enteritidis.

Salmonella Enteritidis (SE) causes both horizontal and vertical transmission of diseases in poultry industry and is also one of the main causes of human food poisoning. Sequence analysis of the sef operon of poultry-derived Salmonella serotypes showed the presence of an entire sef operon in SE, whereas only sef pseudogenes were found in Salmonella Gallinarum and Salmonella Pullorum. Subsequently, the sef operon of SE was cloned into the pBR322 plasmid and expressed in a modified Escherichia coli strain SE5000. sef operon expression was demonstrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, agglutination assay, and transmission electron microscopy. The results showed that SE5000+Sef, but not SE5000+pBR322, could specifically react with SE-positive chicken serum in an agglutination assay, which could be clearly visualized by the naked eye within less than 2 min. In contrast, SE5000+Sef could not be recognized in Salmonella Gallinarum- and Salmonella Pullorum-positive chicken sera. Next, taking advantage of the exclusive presence of an entire sef operon in SE, we set up an agglutination-based detection system to monitor the dynamics of Sef-targeted antibody from SE-infected chicks for 47 days. Using the proposed detection method, SE was readily detectable starting from 2 weeks post-infection. Finally, we compared the proposed SE5000+Sef-based detection system with commercially available agglutination antigen using the classical bacterial isolation and identification procedure as reference. The results showed that the SE5000+Sef system was more consistent with the results of bacterial isolation and identification with almost 100% accuracy. We established a simple, sensitive, and cheap agglutination method for rapid and specific detection of SE-infected chickens, which can facilitate epidemiological investigation and eradication of SE infections. KEY POINTS: • Only the Salmonella Enteritidis serotype expressed Sef fimbriae in chicken infected with SE. • A rapid, large-scale method of detection by the naked eye of detection of SE-infected chicken is presented.

Research progress on Toll-like receptor signal transduction and its roles in antimicrobial immune responses.

When microorganisms invade a host, the innate immune system first recognizes the pathogen-associated molecular patterns of these microorganisms through pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are known transmembrane PRRs existing in both invertebrates and vertebrates. Upon ligand recognition, TLRs initiate a cascade of signaling events; promote the pro-inflammatory cytokine, type I interferon, and chemokine expression; and play an essential role in the modulation of the host's innate and adaptive immunity. Therefore, it is of great significance to improve our understanding of antimicrobial immune responses by studying the role of TLRs and their signal molecules in the host's defense against invading microbes. This paper aims to summarize the specificity of TLRs in recognition of conserved microbial components, such as lipoprotein, lipopolysaccharide, flagella, endosomal nucleic acids, and other bioactive metabolites derived from microbes. This set of interactions helps to elucidate the immunomodulatory effect of TLRs and the signal transduction changes involved in the infectious process and provide a novel therapeutic strategy to combat microbial infections.

Zinc uptake system ZnuACB is essential for maintaining pathogenic phenotype of F4ac+ enterotoxigenic E. coli (ETEC) under a zinc restricted environment.

Zinc is the second trace element of living organisms after iron. Given its crucial importance, mammalian hosts restrict the bioavailability of Zinc ions (Zn2+) to bacterial pathogens. As a countermeasure, pathogens utilize high affinity Zn2+ transporters, such as ZnuACB to compete with the host for zinc. It is essential for bacteria to maintain zinc homeostasis and thus maintain their physiology and pathogenesis. In an attempt to uncover the zinc transporter in F4+ enterotoxigenic E. coli (ETEC) C83902, we analyzed two RNA-seq data sets of bacteria samples when different zinc treatments (restriction or abundance) were applied. Considering data revealing that the high affinity zinc uptake system ZnuACB acts as the main transporter in ETEC C83902 to resist zinc deficiency, we deleted znuACB genes to study the role of them in ETEC C83902. The deletion of znuACB genes results in growth perturbation and a sharp decrease in the ability of biofilm formation and adhesion of bacteria in vitro. Taking the data together, this study demonstrates that the ZnuACB system is required for ETEC C83902 to acquire zinc, which highly contributes to ETEC pathogenicity as well.

Rapid detection of flagellated and non-flagellated Salmonella by targeting the common flagellar hook gene flgE.

Salmonella spp. can cause animal and human salmonellosis. In this study, we established a simple method to detect all Salmonella species by amplifying a specific region within the flgE gene encoding the flagellar hook protein. Our preliminary sequence analysis among flagella-associated genes of Salmonella revealed that although Salmonella Gallinarum and Salmonella Pullorum are lacking flagella, they did have flagella-associated genes, including flgE. To investigate in detail, a comparative flgE sequence analysis was conducted using different bacterial strains including flagellated and non-flagellated Salmonella as well as non-Salmonella strains. Two unique regions (481-529 bp and 721-775 bp of the reference sequence) within the flgE open reading frame were found to be highly conserved and specific to all Salmonella species. Next, we designed a pair of PCR primers (flgE-UP and flgE-LO) targeting the above two regions, and performed a flgE-tailored PCR using as template DNA prepared from a total of 76 bacterial strains (31 flagellated Salmonella strains, 26 non-flagellated Salmonella strains, and 19 other non-Salmonella bacteria strains). Results showed that specific positive bands with expected size were obtained from all Salmonella (including flagellated and non-flagellated Salmonella) strains, while no specific product was generated from non-Salmonella bacterial strains. PCR products from the positive bands were confirmed by DNA sequencing. The minimum detection amount for genomic DNA and bacteria cells reached 18.3 pg/µL and 100 colony-forming unit (CFU) per PCR reaction, respectively. Using the flgE-PCR method to detect Salmonella in artificially contaminated milk samples, as low as 1 CFU/mL Salmonella was detectable after an 8-h pre-culture. Meanwhile, the flgE-tailored PCR method was applied to evaluate 247 clinical samples infected with Salmonella from different chicken breeding farms. The detection results indicated that flgE-PCR could be used to specifically detect Salmonella in concordance with the traditional bacterial culture-based detection method. It is worthwhile noticed that identification results using flgE-tailored PCR should be completed within less than 1 day, expanding the result of much faster than the standard method, which took more than 5 days. Overall, the flgE-tailored PCR method can specifically detect flagellated and non-flagellated Salmonella and can serve as a powerful tool for rapid, simple, and sensitive detection of Salmonella species. KEY POINTS : • Targeting flgE gene for all Salmonella spp. found. • The established PCR assay is used to specifically detect all Salmonella spp. • The PCR method is applied to detect clinical Salmonella spp. samples within less than 1 day.

Fimbriae and related receptors for Salmonella Enteritidis.

Infection with Salmonella Enteritidis (SE) is one of the main causes for food- and water-borne diseases, and is a major concern to public health for both humans and animals worldwide. Some fimbrial antigens expressed by SE strains have been described and characterized, containing SEF14, SEF17, SEF21, long polar fimbriae and plasmid-encoded fimbriae, they play a role in bacterial survival in the host or external environment. However, their functions remain to be well elucidated, with the initial attachment and binding for fimbriae-mediated SE infections only minimally understood. Meanwhile, host-pathogen interactions provide insights into receptor modulation of the host innate immune system. Therefore, to well understand the pathogenicity of SE bacteria and to comprehend the host response to infection, the host cell-SE interactions need to be characterized. This review describes SE fimbriae receptors with an emphasis on the interaction between the receptor and SE fimbriae.

Binding determinants in the interplay between porcine aminopeptidase N and enterotoxigenic Escherichia coli F4 fimbriae.

The binding of F4+ enterotoxigenic Escherichia coli (ETEC) and the specific receptor on porcine intestinal epithelial cells is the initial step in F4+ ETEC infection. Porcine aminopeptidase N (APN) is a newly discovered receptor for F4 fimbriae that binds directly to FaeG adhesin, which is the major subunit of the F4 fimbriae variants F4ab, F4ac, and F4ad. We used overlapping peptide assays to map the APN-FaeG binding sites, which has facilitated in the identifying the APN-binding amino acids that are located in the same region of FaeG variants, thereby limiting the major binding regions of APN to 13 peptides. To determine the core sequence motif, a panel of FaeG peptides with point mutations and FaeG mutants were constructed. Pull-down and binding reactivity assays using piglet intestines determined that the amino acids G159 of F4ab, N209 and L212 of F4ac, and A200 of F4ad were the critical residues for APN binding of FaeG. We further show using ELISA and confocal microscopy assay that amino acids 553-568, and 652-670 of the APN comprise the linear epitope for FaeG binding in all three F4 fimbriae variants.

The different roles of hcp1 and hcp2 of the type VI secretion system in Escherichia coli strain CE129.

Type VI secretion system (T6SS) is a secretory system found in Gram-negative bacteria. One of the main structures for T6SS is Hcp (hemolysin co-regulation protein) pipeline. To investigate the role of Hcp major sub-unit genes hcp1 and hcp2 , we deleted hcp1 and hcp2 genes for constructing the in-frame gene deletion mutants. The properties of biofilm formation and the adhesion to chicken embryo fibroblasts cells (DF1 cells) were reduced in the hcp2 mutant. The knockout of hcp1 and hcp2 genes reduced the ability of the avian pathogenic Escherichia coli (APEC) strain CE129 to infect developing chicken embryos. The expression of quorum sensing (QS)-associated genes luxS, lsrR, and pfs were down-regulated in the hcp1 mutant, and the expression of type 1 fimbriae gene fimA and the adhesion-related genes fimC and papC were decreased in the hcp2 mutant, as well as the expression of anti-serum survival factor genes ompA and iss were inhibited in both hcp1 and hcp2 mutants. These results described above from this study help to further elaborate the role of HCP in APEC.

RNA sensors of the innate immune system and their detection of pathogens.

The innate immune system plays a critical role in pathogen recognition and initiation of protective immune response through the recognition of pathogen associated molecular patterns (PAMPs) by its pattern recognition receptors (PRRs). Nucleic acids including RNA and DNA have been recognized as very important PAMPs of pathogens especially for viruses. RNA are the major PAMPs of RNA viruses, to which most severe disease causing viruses belong thus posing a tougher challenge to human and animal health. Therefore, the understanding of the immune biology of RNA PRRs is critical for control of pathogen infections especially for RNA virus infections. RNA PRRs are comprised of TLR3, TLR7, TLR8, RIG-I, MDA5, NLRP3, NOD2, and some other minorities. This review introduces these RNA PRRs by describing the cellular localizations, ligand recognitions, activation mechanisms, cell signaling pathways, and recognition of pathogens; the cross-talks between various RNA PRRs are also reviewed. The deep insights of these RNA PRRs can be utilized to improve anti-viral immune response. © 2017 IUBMB Life, 69(5):297-304, 2017.

The flagellin hypervariable region is a potential flagella display domain in probiotic Escherichia coli strain Nissle 1917.

The most studied probiotic, Escherichia coli strain Nissle 1917 (EcN) possesses flagella of serotype H1. To explore the potential to use EcN flagellin in flagella display applications, we investigated the effect of deleting amino acids in the hypervariable region of flagellin on EcNc (EcN cured of its two cryptic plasmids pMUT1 and pMUT2). Two EcNc flagellin isogenic mutants with deletions of amino acid residual from 277 to 286 and from 287 to 296 in the hypervariable domain were constructed. Both mutants were flagellated, adherent to IPEC-J2 cells, and colonized BALB/c mice. These hypervariable regions may have future utility in the display of heterologous epitopes.

Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.

F4(+) enterotoxigenic Escherichia coli (ETEC) strains cause diarrheal disease in neonatal and post-weaned piglets. Several different host receptors for F4 fimbriae have been described, with porcine aminopeptidase N (APN) reported most recently. The FaeG subunit is essential for the binding of the three F4 variants to host cells. Here we show in both yeast two-hybrid and pulldown assays that APN binds directly to FaeG, the major subunit of F4 fimbriae, from three serotypes of F4(+) ETEC. Modulating APN gene expression in IPEC-J2 cells affected ETEC adherence. Antibodies raised against APN or F4 fimbriae both reduced ETEC adherence. Thus, APN mediates the attachment of F4(+) E. coli to intestinal epithelial cells.

[Advances in new vaccines against human enterotoxigenic Escherichia coli--A review].

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea, which is a second leading cause of death for the children under five years old from all over the world. The key factors of ETEC contain both colonization factors (CFs) and enterotoxins including heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). CFs mediated the binding of bacteria to the host intestinal epithelial cells, whereas LT and ST stimulated the over-secretion of body fluids and electrolytes, resulting in the destruction of the host fluid balance and leading diarrhea. The vaccine against CFs and enterotoxins could stimulate the host immune response, blocking ETEC adhesion and neutralizing enterotoxins, which is effective in the prevention of ETEC diarrhea. For the moment, depending on the stimulated immune response against LT, a cholera vaccine called Dukoral has been approved for use in some countries for the short-term protection and prevention of travelers' diarrhea. ETEC candidate vaccines are still in progress, which is designed to provide a long and wide-spectrum protection for ETEC infections. This paper briefly summarizes the advanced findings and key problems of vaccine development, and discusses prospects for future research.

Receptor for the F4 fimbriae of enterotoxigenic Escherichia coli (ETEC).

Infection with F4(+) enterotoxigenic Escherichia coli (ETEC) responsible for diarrhea in neonatal and post-weaned piglets leads to great economic losses in the swine industry. These pathogenic bacteria express either of three fimbrial variants F4ab, F4ac, and F4ad, which have long been known for their importance in host infection and initiating protective immune responses. The initial step in infection for the bacterium is to adhere to host enterocytes through fimbriae-mediated recognition of receptors on the host cell surface. A number of receptors for ETEC F4 have now been described and characterized, but their functions are still poorly understood. The current review summarizes the latest research addressing the characteristics of F4 fimbriae receptors and the interactions of F4 fimbriae and their receptors on host cells. These include observations that as follows: (1) FaeG mediates the binding activities of F4 and is an essential component of the F4 fimbriae, (2) the F4 fimbrial receptor gene is located in a region of chromosome 13, (3) the biochemical properties of F4 fimbrial receptors that form the binding site of the bacterium are now recognized, and (4) specific receptors confer susceptibility/resistance to ETEC F4 infection in pigs. Characterizing the host-pathogen interaction will be crucial to understand the pathogenicity of the bacteria, provide insights into receptor activation of the innate immune system, and develop therapeutic strategies to prevent this illness.