Comparative genomic analysis of pathogenic factors of Listeria spp. using whole-genome sequencing.

Listeria monocytogenes is an important foodborne pathogen known for causing listeriosis. To gain insights into the pathogenicity, genetic characterization, and evolution of various Listeria species, in vitro cell adhesion and invasion ability assays and whole-genome sequencing were performed using four Listeria strains isolated from livestock and poultry slaughterhouses. The four Listeria strains exhibited adhesion and invasion abilities in Caco-2 and RAW264.7 cells. Pathogenic Liv1-1 and Lm2-20 had higher adhesion ability, but non-pathogenic Lin4-99 was more invasive than Lm2-20 (p < 0.05). Genetic characterization revealed the presence of a single chromosome without plasmid across four strains with similar whole-genome sizes and G + C% content. Analysis of key pathogenic genes underscored the presence of multiple virulence genes among the four Listeria strains. In contrast, non-pathogenic Listeria lacked LIPI-1, LIPI-2, and LIPI-3 genes, which could possibly be the cause of their non-pathogenicity despite their in vitro cell adhesion and invasion abilities. Thus, genetic determinants of Listeria do not necessarily predict cell adhesion and/or invasive ability in vitro. This study presents a comprehensive comparative genome-wide analysis of four Listeria strains, offering invaluable insights into the pathogenesis of the Listeria genus.

Biofilm-specific determinants of enterococci pathogen.

Amongst all Enterococcus spp., E. faecalis and E. faecium are most known notorious pathogen and their biofilm formation has been associated with endocarditis, oral, urinary tract, and wound infections. Biofilm formation involves a pattern of initial adhesion, microcolony formation, and mature biofilms. The initial adhesion and microcolony formation involve numerous surface adhesins e.g. pili Ebp and polysaccharide Epa. The mature biofilms are maintained by eDNA, It's worth noting that phage-mediated dispersal plays a prominent role. Further, the involvement of peptide pheromones in regulating biofilm maintenance sets it apart from other pathogens and facilitating the horizontal transfer of resistance genes. The role of fsr based regulation by regulating gelE expression is also discussed. Thus, we provide a concise overview of the significant determinants at each stage of Enterococcus spp. biofilm formation. These elements could serve as promising targets for antibiofilm strategies.

AcsS Negatively Regulates the Transcription of type VI Secretion System 2 Genes in Vibrio parahaemolyticus.

The type VI secretion system 2 (T6SS2) gene cluster of Vibrio parahaemolyticus comprises three operons: VPA1027-1024, VPA1043-1028, and VPA1044-1046. AcsS is a LysR-like transcriptional regulator that play a role in activating flagella-driven motility in V. parahaemolyticus. However, its potential roles in other cellular pathways remain poorly understood. In this study, we conducted a series of experiments to investigate the regulatory effects of AcsS on the transcription of VPA1027 (hcp2), VPA1043, and VPA1044. The findings revealed that AcsS indirectly inhibits the transcription of these genes. Additionally, deletion of acsS resulted in enhanced adhesion of V. parahaemolyticus to HeLa cells. However, disruption of T6SS2 alone or in conjunction with AcsS significantly diminished the adhesion capacity of V. parahaemolyticus to HeLa cells. Therefore, it is suggested that AcsS suppresses cell adhesion in V. parahaemolyticus by downregulating the transcription of T6SS2 genes.

The SaeRS two-component system regulates virulence gene expression in group B Streptococcus during invasive infection.

Group B Streptococcus (GBS) is a pathobiont responsible for invasive infections in neonates and the elderly. The transition from a commensal to an invasive pathogen relies on the timely regulation of virulence factors. In this study, we characterized the role of the SaeRS two-component system in GBS pathogenesis. Loss-of-function mutations in the SaeR response regulator decrease virulence in mouse models of invasive infection by hindering the ability of bacteria to persist at the inoculation site and to spread to distant organs. Transcriptome and in vivo analysis reveal a specialized regulatory system specifically activated during infection to control the expression of only two virulence factors: the PbsP adhesin and the BvaP secreted protein. The in vivo surge in SaeRS-regulated genes is complemented by fine-tuning mediated by the repressor of virulence CovRS system to establish a coordinated response. Constitutive activation of the SaeRS regulatory pathway increases PbsP-dependent adhesion and invasion of epithelial and endothelial barriers, though at the cost of reduced virulence. In conclusion, SaeRS is a dynamic, highly specialized regulatory system enabling GBS to express a restricted set of virulence factors that promote invasion of host barriers and allow these bacteria to persist inside the host during lethal infection. IMPORTANCE: Group B Streptococcus (or GBS) is a normal inhabitant of the human gastrointestinal and genital tracts that can also cause deadly infections in newborns and elderly people. The transition from a harmless commensal to a dangerous pathogen relies on the timely expression of bacterial molecules necessary for causing disease. In this study, we characterize the two-component system SaeRS as a key regulator of such virulence factors. Our analysis reveals a specialized regulatory system that is activated only during infection to dynamically adjust the production of two virulence factors involved in interactions with host cells. Overall, our findings highlight the critical role of SaeRS in GBS infections and suggest that targeting this system may be useful for developing new antibacterial drugs.

Expression of the locus of enterocyte effacement genes during the invasion process of the atypical enteropathogenic Escherichia coli 1711-4 strain of serotype O51:H40.

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in low- and middle-income countries. Certain aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. It can also translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of a type III secretion system for the efficiency of the invasion process was demonstrated, the expression of the locus of enterocyte effacement (LEE) genes during the invasion and intracellular persistence remains unclear. To address this question, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro. The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 h post-infection during the persistence period. The number of actin accumulation foci formed on HeLa cells also increased during the 6-h analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that the LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.IMPORTANCEAtypical enteropathogenic Escherichia coli (aEPEC) is a major cause of diarrhea, especially in low- and middle-income countries, like Brazil. However, due to the genome heterogeneity of each clonal group, it is difficult to comprehend the pathogenicity of this strain fully. Among aEPEC strains, 1711-4 can invade eukaryotic cells in vitro, cross the gut barrier, and reach extraintestinal sites in animal models. By studying how different known aEPEC virulence factors are expressed during the invasion process, we can gain insight into the commonalities of this phenotype among other aEPEC strains. This will help in developing preventive measures to control infections caused by invasive strains. No known virulence-encoding genes linked to the invasion process were found. Nevertheless, additional studies are still necessary to evaluate the role of other factors in this phenotype.

The RNA chaperone Hfq has a multifaceted role in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qß replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E. ictaluri by creating an hfq mutant (EiΔhfq) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella. The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, EiΔhfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, EiΔhfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. EiΔhfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the EiΔhfq vaccination completely protected catfish against subsequent EiWT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri, affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.

Tad pili contribute to the virulence and biofilm formation of virulent Aeromonas hydrophila.

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170 A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.

TagP, a PAAR-domain containing protein, plays roles in the fitness and virulence of Acinetobacter baumannii.

Background: Type VI secretion system (T6SS) is widely present in Gram-negative bacteria and directly mediates antagonistic prokaryote interactions. PAAR (proline-alanine-alanine-arginine repeats) proteins have been proven essential for T6SS-mediated secretion and target cell killing. Although PAAR proteins are commonly found in A. baumannii, their biological functions are not fully disclosed yet. In this study, we investigated the functions of a PAAR protein termed TagP (T6SS-associated-gene PAAR), encoded by the gene ACX60_RS09070 outside the core T6SS locus of A. baumannii strain ATCC 17978. Methods: In this study, tagP null and complement A. baumannii ATCC 17978 strains were constructed. The influence of TagP on T6SS function was investigated through Hcp detection and bacterial competition assay; the influence on environmental fitness was studied through in vitro growth, biofilm formation assay, surface motility assay, survivability in various simulated environmental conditions; the influence on pathogenicity was explored through cell adhesion and invasion assays, intramacrophage survival assay, serum survival assay, and G. melonella Killing assays. Quantitative transcriptomic and proteomic analyses were utilized to observe the global impact of TagP on bacterial status. Results: Compared with the wildtype strain, the tagP null mutant was impaired in several tested phenotypes such as surface motility, biofilm formation, tolerance to adverse environments, adherence to eukaryotic cells, endurance to serum complement killing, and virulence to Galleria melonella. Notably, although RNA-Seq and proteomics analysis revealed that many genes were significantly down-regulated in the tagP null mutant compared to the wildtype strain, there is no significant difference in their antagonistic abilities. We also found that Histone-like nucleoid structuring protein (H-NS) was significantly upregulated in the tagP null mutant at both mRNA and protein levels. Conclusions: This study enriches our understanding of the biofunction of PAAR proteins in A. baumannii. The results indicates that TagP involved in a unique modulation of fitness and virulence control in A. baumannii, it is more than a classic PAAR protein involved in T6SS, while how TagP play roles in the fitness and virulence of A. baumannii needs further investigation to clarify.

Genes involved in the adhesion and invasion of Arcobacter butzleri.

Arcobacter butzleri is a foodborne pathogen that mainly causes enteritis in humans, but the number of cases of bacteraemia has increased in recent years. However, there is still limited knowledge on the pathogenic mechanisms of this bacterium. To investigate how A. butzleri causes disease, single knockout mutants were constructed in the cadF, ABU_RS00335, ciaB, and flaAB genes, which might be involved in adhesion and invasion properties. These mutants and the isogenic wild-type (WT) were then tested for their ability to adhere and invade human Caco-2 and HT29-MTX cells. The adhesion and invasion of A. butzleri RM4018 strain was also visualized by a Leica CTR 6500 confocal microscope. The adhesion and invasion abilities of mutants lacking the invasion antigen CiaB or a functional flagellum were lower than those of the WTs. However, the extent of the decrease varied depending on the strain and/or cell line. Mutants lacking the fibronectin (FN)-binding protein CadF consistently exhibited reduced abilities, while the inactivation of the other studied FN-binding protein, ABU_RS00335, led to a reduction in only one of the two strains tested. Therefore, the ciaB and flaAB genes appear to be important for A. butzleri adhesion and invasion properties, while cadF appears to be indispensable.

Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

The role of DsbA and PepP genes in the environmental tolerance and virulence factors of Cronobacter sakazakii.

Cronobacter sakazakii, an opportunity foodborne pathogen, could contaminate a broad range of food materials and cause life-threatening symptoms in infants. The bacterial envelope structure contribute to bacterial environment tolerance, biofilm formation and virulence in various in Gram-negative bacteria. DsbA and PepP are two important genes related to the biogenesis and stability of bacterial envelope. In this study, the DsbA and PepP were deleted in C. sakazakii to evaluate their contribution to stress tolerance and virulence of the pathogen. The bacterial environment resistance assays showed DsbA and PepP are essential in controlling C. sakazakii resistance to heat and desiccation in different mediums, as well as acid, osmotic, oxidation and bile salt stresses. DsbA and PepP also played an important role in regulating biofilm formation and motility. Furthermore, DsbA and PepP deletion weaken C. sakazakii adhesion and invasion in Caco-2, intracellular survival and replication in RAW 264.7. qRT-PCR results showed that DsbA and PepP of C. sakazakii played roles in regulating the expression of several genes associated with environment stress tolerance, biofilm formation, bacterial motility and cellular invasion. These findings indicate that DsbA and PepP played an important regulatory role in the environment resisitance, biofilm formation and virulence of C. sakazakii, which enrich understanding of genetic determinants of adaptability and virulence of the pathogen.

Probiotic functional gene explorations in the genome of Limosilactobacillus fermentum GD5MG.

Limosilactobacillus fermentum is an isolate obtained from oral gingival samples of healthy human individuals. The whole genome of Lb. fermentum GD5MG is composed of a circular DNA molecule containing 1,834,134 bp and exhibits a GC content of 52.80 %. The sequencing effort produced 38.6 million reads, each 150 bp in length, resulting in a sequencing depth of 2912.48x. Our examination unveiled a total of 1961 protein-coding genes, 27 rRNA genes, 24 tRNA genes, 3 non-coding RNA genes, and 63 pseudogenes with the use of gene annotations in NCBI Prokaryotic Genome Annotation tool. RAST revealed 1863 coding genes distributed across 209 subsystems, with a predominant involvement in amino acid, carbohydrate, and protein metabolism. Phylogenetic analysis infers that the Lb. fermentum GD5MG shares 281 gene clusters. Furthermore, the genome features showed a single CRISPR locus of 45 bp in length. Three genes associated with adhesion ability (strA, dltD, and dltA) and 26 genes related to acid tolerance, digestive enzyme secretion, and bile salt resistance were identified. Numerous genes associated with oral probiotic properties, comprising adhesion, acid and bile salt tolerance, oxidative stress tolerance, and sugar metabolism, were identified in the genome. Our findings shed light on the genomic characteristics of Lb. fermentum GD5MG, which are probable probiotics with functional benefits in humans.

The CpxRA two-component system of adherent and invasive Escherichia coli contributes to epithelial cell invasion and early-stage intestinal fitness in a dysbiotic mouse model mediated by type 1 fimbriae expression.

Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn's disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn's disease.

Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene deletion mutant.

Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.

The type IVc pilus: just a Tad different.

Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require multisubunit, membrane-spanning nanomachines for assembly. The tight adherence (Tad) pili are an Archaea-derived T4P subgroup whose machinery exhibits significant mechanistic and architectural differences from bacterial type IVa and IVb pili. Most Tad biosynthetic genes are encoded in a single locus that is widespread in bacteria due to facile acquisition via horizontal gene transfer. These loci experience extensive structural rearrangements, including the acquisition of novel regulatory or biosynthetic genes, which fine-tune their function. This has permitted their integration into many different bacterial lifestyles, including the Caulobacter crescentus cell cycle, Myxococcus xanthus predation, and numerous plant and mammalian pathogens and symbionts.

It takes two to attach - endo-1,3-ß-d-glucanase as a potential receptor of mannose-independent, FimH-dependent Salmonella Typhimurium binding to spinach leaves.

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-ß-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Functional characterization and transcriptional analysis of degQ of Xanthomonas campestris pathovar campestris.

High-temperature-requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone-like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen.

Characterization of clumpy adhesion of Escherichia coli to human cells and associated factors influencing antibiotic sensitivity.

Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. IMPORTANCE: The study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.

Butyrate reduces adherent-invasive E. coli-evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3.

Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn's disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1α mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate's effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.

Response mechanisms to acid stress promote LF82 replication in macrophages.

Background: Adherent-invasive E. coli (AIEC) LF82 is capable of adhering to and invading intestinal epithelial cells, as well as replicating within macrophages without inducing host cell death. Methods: We compared the transcriptomics of LF82 at pH=7.5 and pH=5.8 by RNA-sequencing, and qRT-PCR verified differentially expressed genes (DEGs). The deletion mutants of DEGs in the treatment group (pH=5.8) compared to the control group (pH=7.5) were constructed by λ recombinant. The replication differences between the mutants and WT infected Raw 264.7 at 24 h.p.i were analyzed by combining LB solid plate count and confocal observation. NH4Cl and chloroquine diphosphate (CQ) were used for acid neutralization to study the effect of pH on the replication of LF82 in macrophages. Na2NO3 was added to RPMI 1640 to study the effect of nitrate on the replication of LF82 in macrophages. 0.3% solid LB was used for flagellar motility assay and Hela was used to study flagellar gene deletion mutants and WT adhesion and invasion ability. Results: In this study, we found that infection with LF82 results in acidification of macrophages. Subsequent experiments demonstrated that an intracellular acidic environment is necessary for LF82 replication. Transcriptome and phenotypic analysis showed that high expression of acid shock genes and acid fitness genes promotes LF82 replication in macrophages. Further, we found that the replication of LF82 in macrophages was increased under nitrate treatment, and nitrogen metabolism genes of LF82 were upregulated in acid treatment. The replication in macrophages of ΔnarK, ΔnarXL, ΔnarP, and Δhmp were decreased. In addition, we found that the expression of flagellar genes was downregulated in acidic pH and after LF82 invading macrophages. Motility assay shows that the movement of LF82 on an acidic semisolid agar plate was limited. Further results showed that ΔfliC and ΔfliD decreased in motility, adhesion ability, and invasion of host cells, but no significant effect on replication in macrophages was observed. Conclusion: In this study, we simulated the acidic environment in macrophages, combined with transcriptome technology, and explained from the genetic level that LF82 promotes replication by activating its acid shock and fitness system, enhancing nitrate utilization, and inhibiting flagellar function.