FoxR is an AraC-like transcriptional regulator of ferrioxamine uptake in Salmonella enterica.

Salmonella enterica spp. produce siderophores to bind iron with high affinity and can also use three xenosiderophores secreted by other microorganisms, ferrichrome, coprogen, and ferrioxamine. Here we focused on FoxA, a TonB-dependent transporter of ferrioxamines. Adjacent to foxA is a gene annotated as a helix-turn-helix (HTH) domain-containing protein, SL0358 (foxR), in the Salmonella enterica serovar Typhimurium SL1344 genome. FoxR shares homology with transcriptional regulators belonging to the AraC/XylS family. foxR is syntenic with foxA in the Enterobacteriaceae family, suggesting their functional relatedness. Both foxA and foxR are repressed by the ferric uptake regulator (Fur) under iron-rich growth conditions. When iron is scarce, FoxR acts as a transcriptional activator of foxA by directly binding to its upstream regulatory region. A point mutation in the HTH domain of FoxR abolished this binding, as did mutation of a direct repeat motif in the foxA upstream regulatory region. Desferrioxamine (DFOE) enhanced FoxR protein stability and foxA transcription but did not affect the affinity of FoxR binding to the foxA regulatory region. In summary, we have identified FoxR as a new member of the AraC/XylS family that regulates xenosiderophore-mediated iron uptake by S. Typhimurium and likely other Enterobacteriaceae members.

Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

The Assembly of Flagella in Enteropathogenic Escherichia coli Requires the Presence of a Functional Type III Secretion System.

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco's minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.

The EcpD Tip Adhesin of the Escherichia coli Common Pilus Mediates Binding of Enteropathogenic E. coli to Extracellular Matrix Proteins.

The attachment of enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells is facilitated by several adhesins; however, the individual host-cell receptors for pili-mediated adherence have not been fully characterized. In this study, we evaluated the hypothesis that the E. coli common pilus (ECP) tip adhesin protein EcpD mediates attachment of EPEC to several extracellular matrix (ECM) glycoproteins (fibronectin, laminin, collagens I and IV, and mucin). We found that the ΔecpA mutant, which lacks production of the EcpA filament but retains EcpD on the surface, adhered to these glycoproteins below the wild-type levels, while the ΔecpD mutant, which does not display EcpA or EcpD, bound significantly less to these host glycoproteins. In agreement, a purified recombinant EcpD subunit bound significantly more than EcpA to laminin, fibronectin, collagens I and IV, and mucin in a dose-dependent manner. These are compelling data that strongly suggest that ECP-producing EPEC may bind to host ECM glycoproteins and mucins through the tip adhesin protein EcpD. This study highlights the versatility of EPEC to bind to different host proteins and suggests that the interaction of ECP with the host's ECM glycoproteins may facilitate colonization of the intestinal mucosal epithelium.

Isolation and Identification of Two Brucella Species from a Volcanic Lake in Mexico.

Brucellosis is a zoonosis caused by bacteria of the Brucella genus. Any source of contamination that could be infectious must be monitored to reduce the risk of exposure to brucellosis, so the purpose of this work was to determine the presence of Brucella spp. on surface water and tilapia (Oreochromis niloticus) skin from a volcanic lake in Mexico. A seasonal sampling during 2016-2017 was carried out at fifteen specific sites for water sampling and five sites for the collection of tilapia fish. From all water and fish samples tested, we found only three isolates of Brucella species. We isolated and identified B. abortus from surface water through bacteriological and molecular techniques, and B. abortus and B. suis from the same tilapia skin sample. The isolated strains likely came from breeding animals that are common to the region, such as infected pigs or cattle with Brucella abortus or B. suis, respectively. A similar finding has not been reported in a water from volcanic lake or tilapia fish in Mexico. We concluded that B. abortus and B. suis are present on the surface water of the volcanic lake and tilapia skin as possible contaminants derived from biological material from cows and pigs carrying this bacterium.

Recombinant Escherichia coli BL21 with LngA Variants from ETEC E9034A Promotes Adherence to HT-29 Cells.

The CS21 pilus produced by enterotoxigenic Escherichia coli (ETEC) is involved in adherence to HT-29 intestinal cells. The CS21 pilus assembles proteins encoded by 14 genes clustered into the lng operon. AIM: This study aimed to determine whether E. coli BL21 (ECBL) transformed with the lng operon lacking the lngA gene (pE9034AΔlngA) and complemented in trans with lngA variants of ETEC clinical strains, as well as point substitutions, exhibited modified adherence to HT-29 cells. METHODS: A kanamycin cassette was used to replace the lngA gene in the lng operon of the E9034A strain, and the construct was transformed into the ECBL strain. The pJET1.2 vector carrying lngA genes with allelic variants was transformed into ECBLpE9034AΔlngA (ECBLΔlngA). The point substitutions were performed in the pJETlngAFMU073332 vector. RESULTS: Bioinformatic alignment analysis of the LngA proteins showed hypervariable regions and clustered the clinical ETEC strains into three groups. Variations in amino acid residues affect the adherence percentages of recombinant ECBL strains with lngA variants and site-specific mutations with HT-29 cells. CONCLUSION: In this study, ECBL carrying the lng operon harboring lngA variants of six clinical ETEC strains, as well as point substitutions, exerted an effect on the adherence of ECBL to HT-29 cells, thereby confirming the importance of the CS21 pilus in adherence.

Frequency of respiratory virus-associated infection among children and adolescents from a tertiary-care hospital in Mexico City.

Acute respiratory infections (ARIs) are a major cause of morbidity and mortality among children. The causative pathogens show geographic and seasonal variations. We retrospectively evaluated the frequency and seasonality of respiratory pathogens in children and adolescents (age: 0-19 years) with ARIs treated between January 1, 2021, and March 31, 2022, at a single center in Mexico. Out of 2400 patients, 1,603 were diagnosed with SARS-CoV-2 infection and 797 were diagnosed with other common respiratory pathogens (CRPs). Of the 797 patients, 632 were infected with one CRP and 165 with > 2 CRPs. Deaths occurred only in SARS-CoV-2-infected patients. Rhinovirus/Enterovirus, respiratory syncytial virus B, and parainfluenza virus 3 were the most prevalent in cases with single and multiple infections. CRP showed a high frequency between autumn and winter of 2021, with higher incidence of hospitalization compared to COVID-19. The main comorbidities were immunosuppression, cardiovascular disease (CD), and asthma. The frequency of CRPs showed a downward trend throughout the first half of 2021. CRPs increased in single- and co-infection cases between the fourth and fifth waves of COVID-19, probably due to decreased nonpharmaceutical interventions and changes in diagnostic tests. Age, cyanosis (symptom), and immunosuppression (comorbidity) were found to differentiate between SARS-CoV-2 infection and CRP infection.

The Transcription of Flagella of Enteropathogenic Escherichia coli O127:H6 Is Activated in Response to Environmental and Nutritional Signals.

The flagella of enteropathogenic Escherichia coli (EPEC) O127:H6 E2348/69 mediate adherence to host proteins and epithelial cells. What environmental and nutritional signals trigger or down-regulate flagella expression in EPEC are largely unknown. In this study, we analyzed the influence of pH, oxygen tension, cationic and anionic salts (including bile salt), carbon and nitrogen sources, and catecholamines on the expression of the flagellin gene (fliC) of E2348/69. We found that sodium bicarbonate, which has been shown to induce the expression of type III secretion effectors, down-regulated flagella expression, explaining why E2348/69 shows reduced motility and flagellation when growing in Dulbecco's Minimal Essential Medium (DMEM). Further, growth under a 5% carbon dioxide atmosphere, in DMEM adjusted to pH 8.2, in M9 minimal medium supplemented with 80 mM glucose or sucrose, and in DMEM containing 150 mM sodium chloride, 0.1% sodium deoxycholate, or 30 µM epinephrine significantly enhanced fliC transcription to different levels in comparison to growth in DMEM alone. When EPEC was grown in the presence of HeLa cells or in supernatants of cultured HeLa cells, high levels (4-fold increase) of fliC transcription were detected in comparison to growth in DMEM alone. Our data suggest that nutritional and host signals that EPEC may encounter in the intestinal niche activate fliC expression in order to favor motility and host colonization.

The Fis Nucleoid Protein Negatively Regulates the Phase Variation fimS Switch of the Type 1 Pilus Operon in Enteropathogenic Escherichia coli.

In Escherichia coli the expression of type 1 pili (T1P) is determined by the site-specific inversion of the fimS ON-OFF switch located immediately upstream of major fimbrial subunit gene fimA. Here we investigated the role of virulence (Ler, GrlR, and GrlA) and global regulators (H-NS, IHF, and Fis) in the regulation of the fimS switch in the human enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69. This strain does not produce detectable T1P and PCR analysis of the fimS switch confirmed that it is locked in the OFF orientation. Among the regulator mutants analyzed, only the ∆fis mutant produced significantly high levels of T1P on its surface and yielded high titers of agglutination of guinea pig erythrocytes. Expression analysis of the fimA, fimB, and fimE promoters using lacZ transcriptional fusions indicated that only PfimA activity is enhanced in the absence of Fis. Collectively, these data demonstrate that Fis is a negative regulator of T1P expression in EPEC and suggest that it is required for the FimE-dependent inversion of the fimS switch from the ON-to-OFF direction. It is possible that a similar mechanism of T1P regulation exists in other intestinal and extra-intestinal pathogenic classes of E. coli.

Clinical Characteristics of Coronavirus Disease (COVID-19) in Mexican Children and Adolescents.

BACKGROUND: We analyzed the demographic, clinical, and diagnostic data of children and adolescents in Mexico, from the first case of coronavirus disease (COVID-19) to 28 February 2022. METHODS: Using the open databases of the Ministry of Health and a tertiary pediatric hospital, we obtained demographic and clinical data from the beginning of the COVID-19 pandemic until 28 February 2022. In addition, quantitative reverse-transcription polymerase chain reaction outputs were used to determine the viral load, and structural protein-based serology was performed to evaluate IgG antibody levels. RESULTS: Of the total 437,832 children and adolescents with COVID-19, 1187 died. Of these patients, 1349 were admitted to the Hospital Infantil de Mexico Federico Gómez, and 11 died. Obesity, asthma, and immunosuppression were the main comorbidities, and fever, cough, and headache were the main symptoms. In this population, many patients have a low viral load and IgG antibody levels. CONCLUSION: During the first 2 years of the COVID-19 pandemic in Mexico, children and adolescents had low incidence and mortality. They are a heterogeneous population, but many patients had comorbidities such as obesity, asthma, and immunosuppression; symptoms such as fever, cough, and headache; and low viral load and IgG antibodies.

Coliform bacteria in San Pedro Lake, western Mexico.

Urbanization, livestock activities, and rainfall are factors that contribute to the contamination of inland water. This study aimed to determine the spatial and temporal variability of total coliforms (TCs) and fecal coliforms (FCs) in the surface water of San Pedro Lake as well as the gills and skin of Nile tilapia (Oreochromis niloticus) cultivated in the lake. The study consisted of seasonal sampling during an annual cycle. Using the multiple-tube fermentation technique, we quantified the microbial load of TCs in the lake and fish. The median of the TC and FC groups in surface water showed differences during the seasonal cycle, in which a significant correlation was observed between rainfall and bacterial load in the lake surface water. There was a significant seasonal difference between FCs and TCs in the gills as well as in skin FCs. Anthropogenic activities in the watershed combined with rainfall influence the bacterial load of San Pedro Lake. However, the water quality is still classified as excellent and uncontaminated according to Mexican regulations with lower FC values acceptable for higher FC values. In addition, the bacterial load in tilapia from San Pedro Lake does not pose a risk to human health. PRACTITIONER POINTS: Watershed livestock activities combined with rainfall increase fecal matter pollution in specific areas of the lake. San Pedro Lake displays satisfactory quality for aquatic life. The median fecal coliform population in lake fish (gills and skin) differs by season.

Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México.

Tracking Bioluminescent ETEC during In vivo BALB/c Mouse Colonization.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea worldwide. Adhesion to the human intestinal tract is crucial for colonization. ETEC adhesive structures have been extensively studied; however, colonization dynamics remain uncharacterized. The aim of this study was to track bioluminescent ETEC during in vivo infection. The promoter region of dnaK was fused with the luc gene, resulting in the pRMkluc vector. E. coli K-12 and ETEC FMU073332 strains were electroporated with pRMkluc. E. coli K-12 pRMkluc was bioluminescent; in contrast, the E. coli K-12 control strain did not emit bioluminescence. The highest light emission was measured at 1.9 OD600 (9 h) and quantified over time. The signal was detected starting at time 0 and up to 12 h. Streptomycin-treated BALB/c mice were orogastrically inoculated with either ETEC FMU073332 pRMkluc or E. coli K-12 pRMkluc (control), and bacterial colonization was determined by measuring bacterial shedding in the feces. ETEC FMU073332 pRMkluc shedding started and stopped after inoculation of the control strain, indicating that mouse intestinal colonization by ETEC FMU073332 pRMkluc lasted longer than colonization by the control. The bioluminescence signal of ETEC FMU073332 pRMkluc was captured starting at the time of inoculation until 12 h after inoculation. The bioluminescent signal emitted by ETEC FMU073332 pRMkluc in the proximal mouse ileum was located, and the control signal was identified in the cecum. The detection of maximal light emission and bioluminescence duration allowed us to follow ETEC during in vivo infection. ETEC showed an enhanced colonization and tropism in the mouse intestine compared with those in the control strain. Here, we report the first study of ETEC colonization in the mouse intestine accompanied by in vivo imaging.

Corrigendum: Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A.

[This corrects the article on p. 1201 in vol. 7, PMID: 27536289.].

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372-398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies generated against FimH, CsgA, PapG, FC, and FCP blocked the adhesion of E. coli strain CFT073 to HTB5 bladder cells. In conclusion, the FC and FCP proteins were highly stable, demonstrated antigenic properties, and induced cytokine release (IL-6 and IL-8); furthermore, antibodies generated against these proteins showed protection against bacterial adhesion.

Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and the adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologs. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy images to show that the LngB protein could be localized at the tip of CS21. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells.