Lipopolysaccharide Modification Research Articles

Virulence and Stress Responses of Shigella flexneri Regulated by PhoP/PhoQ.

The two-component signal transduction system PhoP/PhoQ is an important regulator for stress responses and virulence in most Gram-negative bacteria, but characterization of PhoP/PhoQ in Shigella has not been thoroughly investigated. In the present study, we found that deletion of phoPQ (ΔphoPQ) from Shigella flexneri 2a 301 (Sf301) resulted in a significant decline (reduced by more than 15-fold) in invasion of HeLa cells and Caco-2 cells, and less inflammation (− or +) compared to Sf301 (+++) in the guinea pig Sereny test. In low Mg2+ (10 μM) medium or pH 5 medium, the ΔphoPQ strain exhibited a growth deficiency compared to Sf301. The ΔphoPQ strain was more sensitive than Sf301 to polymyxin B, an important antimicrobial agent for treating multi-resistant Gram-negative infections. By comparing the transcriptional profiles of ΔphoPQ and Sf301 using DNA microarrays, 117 differentially expressed genes (DEGs) were identified, which were involved in Mg2+ transport, lipopolysaccharide modification, acid resistance, bacterial virulence, respiratory, and energy metabolism. Based on the reported PhoP box motif [(T/G) GTTTA-5nt-(T/G) GTTTA], we screened 38 suspected PhoP target operons in S. flexneri, and 11 of them (phoPQ, mgtA, slyB, yoaE, yrbL, icsA, yhiWX, rstA, hdeAB, pagP, and shf–rfbU-virK-msbB2) were demonstrated to be PhoP-regulated genes based on electrophoretic mobility shift assays and β-galactosidase assays. One of these PhoP-regulated genes, icsA, is a well-known virulence factor in S. flexneri. In conclusion, our data suggest that the PhoP/PhoQ system modulates S. flexneri virulence (in an icsA-dependent manner) and stress responses of Mg2+, pH and antibacterial peptides.

Microbial resistance to colistin in consequence of mutations in the MCR-1 gene of Escherichia coli

Objective: To evaluate the current situation of microbial resistance to colistin caused by mutations in the MCR-1 gene in Escherichia coli. Materials and methods: A systematic review of the literature was carried out using scientific papers published in Scielo and Pubmed database since 2000. Results: The plasmid-mediated colistin resistance MCR-1 gene is a transmissible gene that causes lipopolysaccharide modifications by regulation systems, presented on chronological scale to the inactivation of drugs that interact with the phospholipids and disorganize the structure of the cell membranes due to the indiscriminate use of colistin. Microbial resistance is mostly related to chromosomal mutations, however, when it refers to colistin it often occurs through horizontal gene transfer. Conclusion: Whereas the mechanisms of microbial resistance to colistin it was possible to conclude that microbial resistance is mostly related to chromosomal mutations, however, when it refers to colistin it often occurs through horizontal gene transfer by plasmid.

Vaccine development targeting lipopolysaccharide structure modification

Vaccines are one of the most important methods for preventing infectious disease. Structural modification of lipopolysaccharide (LPS) provides a strategy for the development of live attenuated vaccines, either by altering the immunogenicity or by attenuating virulence of the bacteria. This review summarizes various approaches that utilize LPS mutants as whole-cell vaccines.

Vibrio cholerae amino acids go on the defense

Gram-negative bacteria remodel their surfaces to interact with the environment, particularly to protect pathogens from immune surveillance and host defenses. The enzyme AlmG is known to be involved in remodeling the Vibrio cholerae surface, but its specific role was not clear. A new study characterizes AlmG at the molecular level, showing it defies phylogenetic expectations to add amino acids to lipopolysaccharide (LPS). This LPS modification plays a pivotal role in V. cholerae resistance to antimicrobial peptides, weapons of the innate immune system against infections.

AlmG, responsible for polymyxin resistance in pandemic Vibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferases

Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. However, some bacteria have developed resistance mechanisms to survive these compounds. Current pandemic O1 Vibrio cholerae biotype El Tor is resistant to polymyxins, whereas a previous pandemic strain of the biotype Classical is polymyxin-sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to antimicrobial peptides through aminoacylation of lipopolysaccharide (LPS), expected to decrease the negatively charged surface of the V. cholerae outer membrane. This Gram-negative system bears striking resemblance to a related Gram-positive cell-wall remodeling strategy that also promotes CAMP resistance. Mutants defective in AlmEF-dependent LPS modification exhibit reduced fitness in vivo Here, we present investigation of AlmG, the hitherto uncharacterized member of the AlmEFG pathway. Evidence for AlmG glycyl to lipid substrate transferase activity is demonstrated in vivo by heterologous expression of V. cholerae pathway enzymes in a specially engineered Escherichia coli strain. Development of a minimal keto-deoxyoctulosonate (Kdo)-lipid A domain in E. coli was necessary to facilitate chemical structure analysis and to produce a mimetic Kdo-lipid A domain AlmG substrate to that synthesized by V. cholerae. Our biochemical studies support a uniquely nuanced pathway of Gram-negative CAMPs resistance and provide a more detailed description of an enzyme of the pharmacologically relevant lysophosphospholipid acyltransferase (LPLAT) superfamily.

Interplay between Colistin Resistance, Virulence and Fitness in Acinetobacter baumannii.

Acinetobacter baumannii is an important opportunistic nosocomial pathogen often resistant to multiple antibiotics classes. Colistin, an “old” antibiotic, is now considered a last-line treatment option for extremely resistant isolates. In the meantime, resistance to colistin has been reported in clinical A. baumannii strains. Colistin is a cationic peptide that disrupts the outer membrane (OM) of Gram-negative bacteria. Colistin resistance is primarily due to post-translational modification or loss of the lipopolysaccharide (LPS) molecules inserted into the outer leaflet of the OM. LPS modification prevents the binding of polymyxin to the bacterial surface and may lead to alterations in bacterial virulence. Antimicrobial pressure drives the evolution of antimicrobial resistance and resistance is often associated with a reduced bacterial fitness. Therefore, the alterations in LPS may induce changes in the fitness of A. baumannii. However, compensatory mutations in clinical A. baumannii may ameliorate the cost of resistance and may play an important role in the dissemination of colistin-resistant A. baumannii isolates. The focus of this review is to summarize the colistin resistance mechanisms, and understand their impact on the fitness and virulence of bacteria and on the dissemination of colistin-resistant A. baumannii strains.

CXC Chemokines Exhibit Bactericidal Activity against Multidrug-Resistant Gram-Negative Pathogens.

ABSTRACTThe continued rise and spread of antimicrobial resistance among bacterial pathogens pose a serious challenge to global health. Countering antimicrobial-resistant pathogens requires a multifaceted effort that includes the discovery of novel therapeutic approaches. Here, we establish the capacity of the human CXC chemokines CXCL9 and CXCL10 to kill multidrug-resistant Gram-negative bacteria, including New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae and colistin-resistant members of the family Enterobacteriaceae that harbor the mobile colistin resistance protein MCR-1 and thus possess phosphoethanolamine-modified lipid A. Colistin-resistant K. pneumoniae isolates affected by genetic mutation of the PmrA/PmrB two-component system, a chromosomally encoded regulator of lipopolysaccharide modification, and containing 4-amino-4-deoxy-l-arabinose-modified lipid A were also found to be susceptible to chemokine-mediated antimicrobial activity. However, loss of PhoP/PhoQ autoregulatory control, caused by disruption of the gene encoding the negative regulator MgrB, limited the bactericidal effects of CXCL9 and CXCL10 in a variable, strain-specific manner. Cumulatively, these findings provide mechanistic insight into chemokine-mediated antimicrobial activity, highlight disparities amongst determinants of colistin resistance, and suggest that chemokine-mediated bactericidal effects merit additional investigation as a therapeutic avenue for treating infections caused by multidrug-resistant pathogens.

Genomic analysis of in vivo acquired resistance to colistin and rifampicin in Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen in healthcare facilities responsible for nosocomial infections mostly in immunocompromised patients. Colistin resistance is increasingly reported worldwide in A. baumannii. Here we describe the in vivo selection of colistin and rifampicin resistance in carbapenem-resistant A. baumannii. Antimicrobial susceptibility testing, plasmid analysis and whole-genome sequencing (WGS) were performed to fully characterise the resistome of two clinical isolates (AbS1 and AbS2) selected during treatment. Clinical isolate AbS1 remained susceptible to colistin, rifampicin and tigecycline, whilst AbS2 was susceptible only to tigecycline. PCR analysis revealed the presence of a blaOXA-23-like carbapenemase gene. Kieser extraction revealed an ca. 74 kb plasmid harbouring blaOXA-23. WGS revealed genomes of 3.8 Mbp in size with a G + C content of 38.9%, and both belonged to ST281 according to the Oxford MLST scheme and ST641 according to the Institut Pasteur scheme. The resistome was also composed of naturally occurring β-lactamases, i.e. ADC-25 cephalosporinase and OXA-82 oxacillinase, aminoglycoside resistance genes [aac(3)-Ia, aadA1 and aph(3')-VIa (aphA6)], and mutations in DNA gyrases explaining fluoroquinolone resistance. Single nucleotide polymorphism analysis revealed that both isolates were identical except for a 30-nucleotide duplication within the pmrB gene and a point mutation in the rpoB gene resulting in colistin and rifampicin resistance, respectively. This study highlights the genomic plasticity of A. baumannii under antibiotic pressure. The 10-amino acid duplication in PmrB affects colistin susceptibility by regulating lipopolysaccharide modification through the PmrAB two-component system. These findings provide further information on the molecular mechanisms leading to colistin resistance in A. baumannii.

High-Level Resistance to Colistin Mediated by Various Mutations in the crrB Gene among Carbapenemase-Producing Klebsiella pneumoniae.

Mutations in crrAB genes encoding a two-component regulator involved in modifications of lipopolysaccharide were searched for among a collection of colistin-resistant Klebsiella pneumoniae isolates. Four isolates, respectively, producing carbapenemases NDM-1, OXA-181, or KPC-2 showed mutated CrrB proteins compared with those in wild-type strains. Complementation assays with a wild-type CrrB protein restored the susceptibility to colistin in all cases, confirming the involvement of the identified substitutions in the resistance phenotype.

Novel coordination of lipopolysaccharide modifications in Vibrio cholerae promotes CAMP resistance.

In the environment and during infection, the human intestinal pathogen Vibrio cholerae must overcome noxious compounds that damage the bacterial outer membrane. The El Tor and classical biotypes of O1 V. cholerae show striking differences in their resistance to membrane disrupting cationic antimicrobial peptides (CAMPs), such as polymyxins. The classical biotype is susceptible to CAMPs, but current pandemic El Tor biotype isolates gain CAMP resistance by altering the net charge of their cell surface through glycine modification of lipid A. Here we report a second lipid A modification mechanism that only functions in the V. cholerae El Tor biotype. We identify a functional EptA ortholog responsible for the transfer of the amino-residue phosphoethanolamine (pEtN) to the lipid A of V. cholerae El Tor that is not functional in the classical biotype. We previously reported that mildly acidic growth conditions (pH 5.8) downregulate expression of genes encoding the glycine modification machinery. In this report, growth at pH 5.8 increases expression of eptA with concomitant pEtN modification suggesting coordinated regulation of these LPS modification systems. Similarly, efficient pEtN lipid A substitution is seen in the absence of lipid A glycinylation. We further demonstrate EptA orthologs from non-cholerae Vibrio species are functional.

Polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: An update.

Emerging multidrug-resistant (MDR) nosocomial pathogens are a great threat. Polymyxins, an old class of cationic polypeptide antibiotic, are considered as last-resort drugs in treating infections caused by MDR Gram-negative bacteria. Increased use of polymyxins in treating critically ill patients necessitates routine polymyxin susceptibility testing. However, susceptibility testing both of colistin and polymyxin B (PMB) is challenging. In this review, currently available susceptibility testing methods are briefly discussed. The multicomponent composition of colistin and PMB significantly influences susceptibility testing. In addition, poor diffusion in the agar medium, adsorption to microtitre plates and the synergistic effect of the surfactant polysorbate 80 with polymyxins have a great impact on the performance of susceptibility testing methods This review also describes recently identified chromosomal resistance mechanisms, including modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-l-arabinose (L-Ara4-N) and phosphoethanolamine (pEtN) resulting in alteration of the negative charge, as well as the plasmid-mediated colistin resistance determinants mcr-1, mcr-1.2, mcr-2 and mcr-3.

Polymyxin Resistance in Gram-negative Pathogens.

The polymyxins are one of the last line antimicrobial classes to retain activity against multidrug-resistant Gram-negative bacilli. However, with the increased use of polymyxins in recent years, reports of resistance have also been increasing. We aimed to describe the mechanisms and occurrence of polymyxin resistance in Gram-negative organisms and propose strategies to overcome resistance. The most common mechanism of acquired resistance to the polymyxins is via modification of the bacterial outer membrane lipopolysaccharide. Global epidemiological surveillance studies have reported the occurrence of polymyxin resistance to be most common in Enterobacteriaceae, specifically Enterobacter species and Acinetobacter baumannii. Prevalence of polymyxin-resistant Gram-negative organisms varies significantly by geographical location. Emergence of polymyxin resistance is of great concern, given the limited number of agents available to treat infections caused by multidrug-resistant Gram-negative organisms. Strategies to mitigate the development of polymyxin resistance include dose optimization and using polymyxin agents in combination with other highly active antimicrobial agents.

Structure of O-Antigen and Hybrid Biosynthetic Locus in Burkholderia cenocepacia Clonal Variants Recovered from a Cystic Fibrosis Patient.

Burkholderia cenocepacia is an opportunistic pathogen associated with chronic lung infections and increased risk of death in patients with cystic fibrosis (CF). In this work, we investigated the lipopolysaccharide (LPS) of clinical variants of B. cenocepacia that were collected from a CF patient over a period of 3.5 years, from the onset of infection until death by necrotizing pneumonia (cepacia syndrome). We report the chemical structure of the LPS molecule of various sequential isolates and the identification of a novel hybrid O-antigen (OAg) biosynthetic cluster. The OAg repeating unit of the LPS from IST439, the initial isolate, is a [→2)-β-D-Ribf-(1→4)-α-D-GalpNAc-(1→] disaccharide, which was not previously described in B. cenocepacia. The IST439 OAg biosynthetic gene cluster contains 7 of 23 genes that are closely homologous to genes found in B. multivorans, another member of the Burkholderia cepacia complex. None of the subsequent isolates expressed OAg. Genomic sequencing of these isolates enabled the identification of mutations within the OAg cluster, but none of these mutations could be associated with the loss of OAg. This study provides support to the notion that OAg LPS modifications are an important factor in the adaptation of B. cenocepacia to chronic infection and that the heterogeneity of OAgs relates to variation within the OAg gene cluster, indicating that the gene cluster might have been assembled through multiple horizontal transmission events.

Structural Modification of Lipopolysaccharide Conferred by mcr-1 in Gram-Negative ESKAPE Pathogens.

mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceaemcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.

Two-component systems required for virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of infecting a broad range of hosts, in addition to thriving in a broad range of environmental conditions outside of hosts. With this versatility comes the need to tightly regulate its genome to optimise its gene expression and behaviour to the prevailing conditions. Two-component systems (TCSs) comprising sensor kinases and response regulators play a major role in this regulation. This minireview discusses the growing number of TCSs that have been implicated in the virulence of P. aeruginosa, with a special focus on the emerging theme of multikinase networks, which are networks comprising multiple sensor kinases working together, sensing and integrating multiple signals to decide upon the best response. The networks covered in depth regulate processes such as the switch between acute and chronic virulence (GacS network), the Cup fimbriae (Roc network and Rcs/Pvr network), the aminoarabinose modification of lipopolysaccharide (a network involving the PhoQP and PmrBA TCSs), twitching motility and virulence (a network formed from the Chp chemosensory pathway and the FimS/AlgR TCS), and biofilm formation (Wsp chemosensory pathway). In addition, we highlight the important interfaces between these systems and secondary messenger signals such as cAMP and c-di-GMP.

In vivo Host Environment Alters Pseudomonas aeruginosa Susceptibility to Aminoglycoside Antibiotics

During host infection, Pseudomonas aeruginosa coordinately regulates the expression of numerous genes to adapt to the host environment while counteracting host clearance mechanisms. As infected patients take antibiotics, the invading bacteria encounter antibiotics in the host milieu. P. aeruginosa is highly resistant to antibiotics due to multiple chromosomally encoded resistant determinants. And numerous in vitro studies have demonstrated the regulatory mechanisms of antibiotic resistance related genes in response to antibiotics. However, it is not well-known how host environment affects bacterial response to antibiotics. In this study, we found that P. aeruginosa cells directly isolated from mice lungs displayed higher susceptibility to tobramycin than in vitro cultured bacteria. In vitro experiments demonstrated that incubation with A549 and differentiated HL60 (dHL60) cells sensitized P. aeruginosa to tobramycin. Further studies revealed that reactive oxygen species produced by the host cells contributed to the increased bacterial susceptibility. At the same concentration of tobramycin, presence of A549 and dHL60 cells resulted in higher expression of heat shock proteins, which are known inducible by tobramycin. Further analyses revealed decreased membrane potential upon incubation with the host cells and modification of lipopolysaccharide, which contributed to the increased susceptibility to tobramycin. Therefore, our results demonstrate that contact with host cells increased bacterial susceptibility to tobramycin.

An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence

Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle.

CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease.

Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called “CRISPRs.” Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1 and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD.

Protective Immunity Elicited by Oral Immunization of Mice with Salmonella enterica Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants.

We evaluated the extent of attenuation and immunogenicity of the ΔlppAB and ΔlppAB ΔmsbB mutants of Salmonella enterica serovar Typhimurium when delivered to mice by the oral route. These mutants were deleted either for the Braun lipoprotein genes (lppA and lppB) or in combination with the msbB gene, which encodes an acetyltransferase required for lipid A modification of lipopolysaccharide. Both the mutants were attenuated (100% animal survival) and triggered robust innate and adaptive immune responses. Comparable levels of IgG and its isotypes were produced in mice infected with wild-type (WT) S. typhimurium or its aforementioned mutant strains. The ΔlppAB ΔmsbB mutant-immunized animals resulted in the production of higher levels of fecal IgA and serum cytokines during later stages of vaccination (adaptive response). A significant production of interleukin-6 from T-cells was also noted in the ΔlppAB ΔmsbB mutant-immunized mice when compared to that of the ΔlppAB mutant. On the other hand, IL-17A production was significantly more in the serum of ΔlppAB mutant-immunized mice (innate response) with a stronger splenic T-cell proliferative and tumor-necrosis factor-α production. Based on 2-dimensional gel analysis, alterations in the levels of several proteins were observed in both the mutant strains when compared to that in WT S. typhimurium and could be associated with the higher immunogenicity of the mutants. Finally, both ΔlppAB and ΔlppAB ΔmsbB mutants provided complete protection to immunized mice against a lethal oral challenge dose of WT S. typhimurium. Thus, these mutants may serve as excellent vaccine candidates and also provide a platform for delivering heterologous antigens.

Lipopolysaccharide Domains Modulate Urovirulence.

Uropathogenic Escherichia coli (UPEC) accounts for 80 to 90% of urinary tract infections (UTI), and the increasing rate of antibiotic resistance among UPEC isolates reinforces the need for vaccines to prevent UTIs and recurrent infections. Previous studies have shown that UPEC isolate NU14 suppresses proinflammatory NF-κB-dependent cytokines (D. J. Klumpp, A. C. Weiser, S. Sengupta, S. G. Forrestal, R. A. Batler, and A. J. Schaeffer, Infect Immun 69:6689-6695, 2001, http://dx.doi.org/10.1128/IAI.69.11.6689-6695.2001; B. K. Billips, A. J. Schaeffer, and D. J. Klumpp, Infect Immun 76:3891-3900, 2008, http://dx.doi.org/10.1128/IAI.00069-08). However, modification of lipopolysaccharide (LPS) structure by deleting the O-antigen ligase gene (waaL) enhanced proinflammatory cytokine secretion. Vaccination with the ΔwaaL mutant diminished NU14 reservoirs and protected against subsequent infections. Therefore, we hypothesized that LPS structural determinants shape immune responses. We evaluated the contribution of LPS domains to urovirulence corresponding to the inner core (waaP, waaY, and rfaQ), outer core (rfaG), and O-antigen (waaL, wzzE, and wzyE). Deletion of waaP, waaY, and rfaG attenuated adherence to urothelial cells in vitro In a murine UTI model, the ΔrfaG mutant had the most severe defect in colonization. The mutation of rfaG, waaL, wzzE, and wzyE resulted in an inability to form reservoirs in mouse bladders. Infection with the LPS mutant panel resulted in various levels of urinary myeloperoxidase. Since the ΔwaaL mutant promoted Th1-associated adaptive responses in previous studies (B. K. Billips, R. E. Yaggie, J. P. Cashy, A. J. Schaeffer, and D. J. Klumpp, J Infect Dis 200:263-272, 2009, http://dx.doi.org/10.1086/599839), we assessed NU14 for Th2-associated cytokines. We found NU14 infection stimulated TLR4-dependent bladder interleukin-33 (IL-33) production. Inoculation with rfaG, waaL, wzzE, and wzyE mutants showed decreased IL-33 production. We quantified antigen-specific antibodies after infection and found significantly increased IgE and IgG1 in ΔwaaP mutant-infected mice. Our studies show LPS structural constituents mediate multiple aspects of the UPEC life cycle, including the ability to acutely colonize bladders, form reservoirs, and evoke innate and adaptive immune responses.