Entry into host cells is required for many bacterial pathogens to effectively disseminate within a host, avoid immune detection and cause disease. In recent years, many ostensibly extracellular bacteria have been shown to act as opportunistic intracellular pathogens. Among these are strains of uropathogenic Escherichia coli (UPEC), the primary causative agents of urinary tract infections (UTIs). UPEC are able to transiently invade, survive and multiply within the host cells and tissues constituting the urinary tract. Invasion of host cells by UPEC is promoted independently by distinct virulence factors, including cytotoxic necrotizing factor, Afa/Dr adhesins, and type 1 pili. Here we review the diverse mechanisms and consequences of host cell invasion by UPEC, focusing also on the impact of these processes on the persistence and recurrence of UTIs.

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, α-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.

During the course of a urinary tract infection, substantial levels of nitric oxide and reactive nitrogen intermediates are generated. We have found that many uropathogenic strains of Escherichia coli display far greater resistance to nitrosative stress than the K-12 reference strain MG1655. By selecting and screening for uropathogenic E. coli transposon mutants that are unable to grow in the presence of acidified nitrite, the cadC gene product was identified as a key facilitator of nitrosative stress resistance. Mutation of cadC, or its transcriptional targets cadA and cadB, results in loss of significant production of the polyamine cadaverine and increased sensitivity to acidified nitrite. Exogenous addition of cadaverine or other polyamines rescues growth of cad mutants under nitrosative stress. In wild-type cells, the concentration of cadaverine produced per cell is substantially increased by exposure to acidified nitrite. The mechanism behind polyamine-mediated rescue from nitrosative stress is unclear, but it is not attributable solely to chemical quenching of reactive nitrogen species or reduction in mutation frequency.Along with its roles in neurotransmission, vasodilation, cell adhesion, and platelet inhibition, nitric oxide (NO) is also a key player in innate immunity. NO is formed by the action of nitric oxide synthases (NOS), which are induced and activated during the course of infections caused by many different pathogens. NO may then be converted to a variety of other damaging reactive nitrogen intermediates (RNIs) such as peroxynitrite and nitrosothiols. These RNIs can inflict extensive damage on the nucleic acids, proteins, and lipids of invading microbes. Thiols, amines, aromatic residues, heme groups, and iron-sulfur clusters are particularly susceptible to attack (9, 10). NOS activity has been shown to be crucial in control of pathogen load in numerous infections, particularly Salmonella, Leishmania, and Listeria spp. and mycobacteria (9).Other infections which may be influenced by RNIs are those elicited by strains of uropathogenic Escherichia coli (UPEC). These bacteria are the primary causative agents of urinary tract infections, including both bladder and kidney infections (3). Within hours of initiation of a bladder infection, levels of nitrite in the urine increase threefold (24), and eventually gaseous levels of NO within the bladder increase 30-to 50-fold over uninfected controls (17). Much of this increase is likely due to the action of endothelial (e) NOS in the bladder, which has been shown to be upregulated and activated by intraperitoneal injection of E. coli lipopolysaccharide (14). The bacteria themselves, which can produce NO through nitrite reductases under conditions of low oxygen tension (7), may also contribute to the high levels of RNIs in an infected bladder. Like mycobacteria and several other pathogens that are known to be kept in check by RNIs, UPEC can persist long-term within a host as an intracellular pathogen and may lie in wait in quiescent reservoirs between recur...

In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H 2 O 2 ) and acid stress. In the mutant strains, H 2 O 2 resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract. U nder homeostatic conditions, the mammalian urinary tract is maintained as a sterile environment through the production of antimicrobial peptides and other toxic compounds, the bulk flow of urine, innate immune cell surveillance mechanisms, and nutrient limitations (1-5). However, select microbial pathogens are capable of colonizing and infecting this normally inhospitable niche. Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTI) worldwide, affecting millions and requiring billions of dollars for diagnosis and treatment annually (6). To overcome host defenses and effectively colonize the urinary tract, UPEC employs a variety of virulence factors and stress resistance mechanisms, including adhesive and motility organelles that mediate attachment to and invasion of host cells, toxins that disarm innate immune responses, and multiple iron-scavenging proteins (1, 7-9). The ability to sense and prioritize the use of limited carbon sources within the nutrient-poor environment of the urinary tract is also likely critical to the success of UPEC, but our understanding of the impact that bacterial metabolic pathways have on the establishment and progression of a UTI is incomplete.Within the urinary tract, UPEC relies largely on the catabolism of small peptides and amino acids for survival and growth (4). U...

While in transit within and between hosts, uropathogenic Escherichia coli (UPEC) encounters multiple stresses, including substantial levels of nitric oxide and reactive nitrogen intermediates. Here we show that UPEC, the primary cause of urinary tract infections, can be conditioned to grow at higher rates in the presence of acidified sodium nitrite (ASN), a model system used to generate nitrosative stress. When inoculated into the bladder of a mouse, ASN-conditioned UPEC bacteria are far more likely to establish an infection than nonconditioned bacteria. Microarray analysis of ASN-conditioned bacteria suggests that several NsrR-regulated genes and other stress-and polyamine-responsive factors may be partially responsible for this effect. Compared to K-12 reference strains, most UPEC isolates have increased resistance to ASN, and this resistance can be substantially enhanced by addition of the polyamine cadaverine. Nitrosative stress, as generated by ASN, can stimulate cadaverine synthesis by UPEC, and growth of UPEC in cadaverine-supplemented broth in the absence of ASN can also promote UPEC colonization of the bladder. These results suggest that UPEC interactions with polyamines or stresses such as reactive nitrogen intermediates can in effect reprogram the bacteria, enabling them to better colonize the host.The urinary tract is normally a sterile environment, and it is both hostile and poorly accessible to most microbes. However, roughly one-half of women in the United States experience a urinary tract infection (UTI) at least once in their lifetime, and one-quarter of affected women endure recurrence (22,25). More than 80% of UTIs are due to strains of uropathogenic Escherichia coli (UPEC), which are usually presumed to be part-time gut flora that have reached the urinary tract by ascension via the periurethral area (53). Transmission of UPEC among individuals occurs primarily by way of fecal-oral routes and, in some cases, may involve the ingestion of contaminated food products or sexual contact (15,23,33,40,41,57). In order to survive and disseminate, UPEC must be able to adapt to multiple environments and stresses both within and outside the host.When a UPEC infection occurs, recruitment of nitric oxide (NO)-producing neutrophils to the bladder is an important line of defense (26,48). Within hours of infection, the nitrite levels in the urine increase up to threefold, and eventually the levels of NO within the bladder are 30-to 50-fold higher than those in uninfected controls (39, 48). The high levels of NO are due in part to inducible NO synthase activity, which is upregulated within 6 h after infection (45). A role may also be played by endothelial NO synthase, which is upregulated and activated in the bladder mucosa by E. coli lipopolysaccharide (36) and by the bacteria themselves, which can produce NO with nitrite reductases under low-oxygen-tension conditions (12). NO is a precursor of a variety of reactive nitrogen intermediates (RNIs), such as peroxynitrite and nitrosothiols, which can inflict exten...

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