Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

Transmission of trained immunity and heterologous resistance to infections across generations.

Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.

A TRP Channel Senses Lysosome Neutralization by Pathogens to Trigger Their Expulsion.

Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens.

Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses.

Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.

Predictive phage therapy for Escherichia coli urinary tract infections: Cocktail selection for therapy based on machine learning models.

This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning (ML) models. For this purpose, ML models were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli associated with urinary tract infections. This is an important common infection in humans and companion animals from which multidrug-resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage was measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features, and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive ML models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy.

Glucuronic acid confers colonization advantage to enteric pathogens.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

Insulin receptor orchestrates kidney antibacterial defenses.

Urinary tract infection (UTI) commonly afflicts people with diabetes. This augmented infection risk is partly due to deregulated insulin receptor (IR) signaling in the kidney collecting duct. The collecting duct is composed of intercalated cells (ICs) and principal cells (PCs). Evidence suggests that ICs contribute to UTI defenses. Here, we interrogate how IR deletion in ICs impacts antibacterial defenses against uropathogenic Escherichia coli. We also explore how IR deletion affects immune responses in neighboring PCs with intact IR expression. To accomplish this objective, we profile the transcriptomes of IC and PC populations enriched from kidneys of wild-type and IC-specific IR knock-out mice that have increased UTI susceptibility. Transcriptomic analysis demonstrates that IR deletion suppresses IC-integrated stress responses and innate immune defenses. To define how IR shapes these immune defenses, we employ murine and human kidney cultures. When challenged with bacteria, murine ICs and human kidney cells with deregulated IR signaling cannot engage central components of the integrated stress response-including activating transcriptional factor 4 (ATF4). Silencing ATF4 impairs NFkB activation and promotes infection. In turn, NFkB silencing augments infection and suppresses antimicrobial peptide expression. In diabetic mice and people with diabetes, collecting duct cells show reduced IR expression, impaired integrated stress response engagement, and compromised immunity. Collectively, these translational data illustrate how IR orchestrates collecting duct antibacterial responses and the communication between ICs and PCs.

Host-derived CEACAM-laden vesicles engage enterotoxigenic Escherichia coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here, however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.

The role of bacterial size, shape and surface in macrophage engulfment of uropathogenic E. coli cells.

Uropathogenic Escherichia coli (UPEC) can undergo extensive filamentation in the host during acute urinary tract infections (UTIs). It has been hypothesised that this morphological plasticity allows bacteria to avoid host immune responses such as macrophage engulfment. However, it is still unclear what properties of filaments are important in macrophage-bacteria interactions. The aim of this work was to investigate the contribution of bacterial biophysical parameters, such as cell size and shape, and physiological parameters, such as cell surface and the environment, to macrophage engulfment efficiency. Viable, reversible filaments of known lengths and volumes were produced in the UPEC strain UTI89 using a variety of methods, including exposure to cell-wall targeting antibiotics, genetic manipulation and isolation from an in vitro human bladder cell model. Quantification of the engulfment ability of macrophages using gentamicin-protection assays and fluorescence microscopy demonstrated that the ability of filaments to avoid macrophage engulfment is dependent on a combination of size (length and volume), shape, cell surface and external environmental factors. UTI89 filamentation and macrophage engulfment efficiency were also found to occur independently of the SOS-inducible filamentation genes, sulA and ymfM in both in vivo and in vitro models of infection. Compared to filaments formed via antibiotic inhibition of division, the infection-derived filaments were preferentially targeted by macrophages. With several strains of UPEC now resistant to current antibiotics, our work identifies the importance of bacterial physiological and morphological states during infection.

Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs) in humans. Moreover, as one of the most common bacterial pathogens, UPEC imposes a substantial burden on healthcare systems worldwide. Epithelial cells and macrophages are two major components of the innate immune system, which play critical roles in defending the bladder against UPEC invasion. Yet, the routes of communication between these cells during UTI pathogenesis are still not fully understood. In the present study, we investigated the role of membrane-bound nanovesicles (exosomes) in the communication between bladder epithelial cells and macrophages during UPEC infection, using an array of techniques such as flow cytometry, miRNA profiling, RNA sequencing, and western blotting. Moreover, our in vitro findings were validated in a mouse model of UPEC-induced cystitis. We found that UPEC infection induced the bladder epithelial MB49 cell line to secrete large numbers of exosomes (MB49-U-Exo), which were efficiently absorbed by macrophages both in vivo and in vitro. Assimilation of MB49-U-Exo induced macrophages to produce proinflammatory cytokines, including tumor necrosis factor (TNF)α. Exposure of macrophages to MB49-U-Exo reduced their phagocytic activity (by downregulating the expression of phagocytosis-related genes) and increased their rate of apoptosis. Mechanistically, we showed that MB49-U-Exo were enriched in miR-18a-5p, which induced TNFα expression in macrophages by targeting PTEN and activating the MAPK/JNK signaling pathway. Moreover, administration of the exosome secretion inhibitor GW4869 or a TNFα-neutralizing antibody alleviated UPEC-mediated tissue damage in mice with UPEC-induced cystitis by reducing the bacterial burden of the bladder and dampening the associated inflammatory response. Collectively, these findings suggest that MB49-U-Exo regulate macrophage function in a way that exacerbates UPEC-mediated tissue impairment. Thus, targeting exosomal -release or TNFα signaling during UPEC infection may represent promising non-antibiotic strategies for treating UTIs.

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as volunteers challenged with ETEC, diarrheal severity is significantly increased in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

Microbiota-targeted maternal antibodies protect neonates from enteric infection.

Although maternal antibodies protect newborn babies from infection1,2, little is known about how protective antibodies are induced without prior pathogen exposure. Here we show that neonatal mice that lack the capacity to produce IgG are protected from infection with the enteric pathogen enterotoxigenic Escherichia coli by maternal natural IgG antibodies against the maternal microbiota when antibodies are delivered either across the placenta or through breast milk. By challenging pups that were fostered by either maternal antibody-sufficient or antibody-deficient dams, we found that IgG derived from breast milk was crucial for protection against mucosal disease induced by enterotoxigenic E. coli. IgG also provides protection against systemic infection by E. coli. Pups used the neonatal Fc receptor to transfer IgG from milk into serum. The maternal commensal microbiota can induce antibodies that recognize antigens expressed by enterotoxigenic E. coli and other Enterobacteriaceae species. Induction of maternal antibodies against a commensal Pantoea species confers protection against enterotoxigenic E. coli in pups. This role of the microbiota in eliciting protective antibodies to a specific neonatal pathogen represents an important host defence mechanism against infection in neonates.

A prophage encoded ribosomal RNA methyltransferase regulates the virulence of Shiga-toxin-producing Escherichia coli (STEC).

Shiga toxin (Stx) released by Shiga toxin producing Escherichia coli (STEC) causes life-threatening illness. Its production and release require induction of Stx-encoding prophage resident within the STEC genome. We identified two different STEC strains, PA2 and PA8, bearing Stx-encoding prophage whose sequences primarily differ by the position of an IS629 insertion element, yet differ in their abilities to kill eukaryotic cells and whose prophages differ in their spontaneous induction frequencies. The IS629 element in ϕPA2, disrupts an ORF predicted to encode a DNA adenine methyltransferase, whereas in ϕPA8, this element lies in an intergenic region. Introducing a plasmid expressing the methyltransferase gene product into ϕPA2 bearing-strains increases both the prophage spontaneous induction frequency and virulence to those exhibited by ϕPA8 bearing-strains. However, a plasmid bearing mutations predicted to disrupt the putative active site of the methyltransferase does not complement either of these defects. When complexed with a second protein, the methyltransferase holoenzyme preferentially uses 16S rRNA as a substrate. The second subunit is responsible for directing the preferential methylation of rRNA. Together these findings reveal a previously unrecognized role for rRNA methylation in regulating induction of Stx-encoding prophage.

The bacterial genetic determinants of Escherichia coli capacity to cause bloodstream infections in humans.

Escherichia coli is both a highly prevalent commensal and a major opportunistic pathogen causing bloodstream infections (BSI). A systematic analysis characterizing the genomic determinants of extra-intestinal pathogenic vs. commensal isolates in human populations, which could inform mechanisms of pathogenesis, diagnostic, prevention and treatment is still lacking. We used a collection of 912 BSI and 370 commensal E. coli isolates collected in France over a 17-year period (2000-2017). We compared their pangenomes, genetic backgrounds (phylogroups, STs, O groups), presence of virulence-associated genes (VAGs) and antimicrobial resistance genes, finding significant differences in all comparisons between commensal and BSI isolates. A machine learning linear model trained on all the genetic variants derived from the pangenome and controlling for population structure reveals similar differences in VAGs, discovers new variants associated with pathogenicity (capacity to cause BSI), and accurately classifies BSI vs. commensal strains. Pathogenicity is a highly heritable trait, with up to 69% of the variance explained by bacterial genetic variants. Lastly, complementing our commensal collection with an older collection from 1980, we predict that pathogenicity continuously increased through 1980, 2000, to 2010. Together our findings imply that E. coli exhibit substantial genetic variation contributing to the transition between commensalism and pathogenicity and that this species evolved towards higher pathogenicity.

Distinct ecological fitness factors coordinated by a conserved Escherichia coli regulator during systemic bloodstream infection.

The ability of bacterial pathogens to adapt to host niches is driven by the carriage and regulation of genes that benefit pathogenic lifestyles. Genes that encode virulence or fitness-enhancing factors must be regulated in response to changing host environments to allow rapid response to challenges presented by the host. Furthermore, this process can be controlled by preexisting transcription factors (TFs) that acquire new roles in tailoring regulatory networks, specifically in pathogens. However, the mechanisms underlying this process are poorly understood. The highly conserved Escherichia coli TF YhaJ exhibits distinct genome-binding dynamics and transcriptome control in pathotypes that occupy different host niches, such as uropathogenic E. coli (UPEC). Here, we report that this important regulator is required for UPEC systemic survival during murine bloodstream infection (BSI). This advantage is gained through the coordinated regulation of a small regulon comprised of both virulence and metabolic genes. YhaJ coordinates activation of both Type 1 and F1C fimbriae, as well as biosynthesis of the amino acid tryptophan, by both direct and indirect mechanisms. Deletion of yhaJ or the individual genes under its control leads to attenuated survival during BSI. Furthermore, all three systems are up-regulated in response to signals derived from serum or systemic host tissue, but not urine, suggesting a niche-specific regulatory trigger that enhances UPEC fitness via pleiotropic mechanisms. Collectively, our results identify YhaJ as a pathotype-specific regulatory aide, enhancing the expression of key genes that are collectively required for UPEC bloodstream pathogenesis.

Molecular responses during bacterial filamentation reveal inhibition methods of drug-resistant bacteria.

Bacterial antimicrobial resistance (AMR) is among the most significant challenges to current human society. Exposing bacteria to antibiotics can activate their self-saving responses, e.g., filamentation, leading to the development of bacterial AMR. Understanding the molecular changes during the self-saving responses can reveal new inhibition methods of drug-resistant bacteria. Herein, we used an online microfluidics mass spectrometry system for real-time characterization of metabolic changes of bacteria during filamentation under the stimulus of antibiotics. Significant pathways, e.g., nucleotide metabolism and coenzyme A biosynthesis, correlated to the filamentation of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) were identified. A cyclic dinucleotide, c-di-GMP, which is derived from nucleotide metabolism and reported closely related to bacterial resistance and tolerance, was observed significantly up-regulated during the bacterial filamentation. By using a chemical inhibitor, ebselen, to inhibit diguanylate cyclases which catalyzes the synthesis of c-di-GMP, the minimum inhibitory concentration of ceftriaxone against ESBL-E. coli was significantly decreased. This inhibitory effect was also verified with other ESBL-E. coli strains and other beta-lactam antibiotics, i.e., ampicillin. A mutant strain of ESBL-E. coli by knocking out the dgcM gene was used to demonstrate that the inhibition of the antibiotic resistance to beta-lactams by ebselen was mediated through the inhibition of the diguanylate cyclase DgcM and the modulation of c-di-GMP levels. Our study uncovers the molecular changes during bacterial filamentation and proposes a method to inhibit antibiotic-resistant bacteria by combining traditional antibiotics and chemical inhibitors against the enzymes involved in bacterial self-saving responses.

α-Hemolysin promotes uropathogenic E. coli persistence in bladder epithelial cells via abrogating bacteria-harboring lysosome acidification.

There is a growing consensus that a significant proportion of recurrent urinary tract infections are linked to the persistence of uropathogens within the urinary tract and their re-emergence upon the conclusion of antibiotic treatment. Studies in mice and human have revealed that uropathogenic Escherichia coli (UPEC) can persist in bladder epithelial cells (BECs) even after the apparent resolution of the infection. Here, we found that, following the entry of UPEC into RAB27b+ fusiform vesicles in BECs, some bacteria escaped into the cytoplasmic compartment via a mechanism involving hemolysin A (HlyA). However, these UPEC were immediately recaptured within LC3A/B+ autophagosomes that matured into LAMP1+ autolysosomes. Thereafter, HlyA+ UPEC-containing lysosomes failed to acidify, which is an essential step for bacterial elimination. This lack of acidification was related to the inability of bacteria-harboring compartments to recruit V-ATPase proton pumps, which was attributed to the defragmentation of cytosolic microtubules by HlyA. The persistence of UPEC within LAMP1+ compartments in BECs appears to be directly linked to HlyA. Thus, through intravesicular instillation of microtubule stabilizer, this host defense response can be co-opted to reduce intracellular bacterial burden following UTIs in the bladder potentially preventing recurrence.

Regulation of human neutrophil IL-1ß secretion induced by Escherichia coli O157:H7 responsible for hemolytic uremic syndrome.

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1ß). STEC stimulated neutrophils to release elevated levels of IL-1ß through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1ß secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1ß secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1ß secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation.