Enterohemorrhagic Escherichia coli O157:H7 (EHEC) and enteropathogenic E. coli O127:H7 (EPEC) induce characteristic F-actin rich pedestals on infected mammalian cells. Each pathogen delivers its own translocated intimin receptor (Tir) to the host cell to act as a receptor for the bacterial outer membrane adhesin, intimin. Interaction of translocated Tir with intimin is essential for mammalian cell binding and host colonization, as well as to induce actin pedestal formation in vitro. In spite of these parallels, EHEC and EPEC Tir appear to generate actin pedestals by distinct mechanisms. Further, while the ability to form actin pedestals is a striking phenotype, the function of pedestals during infection remains unclear. To address these issues, a systematic and quantitative analysis of Tir-mediated actin assembly was conducted. We identified a three-residue Tir sequence involved in actin pedestal formation for both EHEC and EPEC, and developed evidence that the two pathogens trigger a common pathway for actin assembly. Further, the ability of these bacteria to promote actin assembly appears to promote both intimin-mediated bacterial binding in vitro and optimal colonization during experimental animal infection.

Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.

Enterohemorrhagic Escherichia coli 0157:H7 (EHEC) and Enteropathogenic E. coli (EPEC) are intestinal pathogens that induce characteristic lesions on mammalian cells called actin pedestals. Attachment to host cells by both EPEC and EHEC is an essential step towards colonization and is associated with the formation of highly organized actin cytoskeletal elements termed as attaching and effacing (AE) lesions beneath bound bacteria. The outer membrane protein intimin is required for the formation of these structures and binds its own translocated mammalian cell receptor called Translocated intimin receptor (Tir). These interactions induce a cascade of events that result in actin pedestal formation. In this thesis, we characterized pedestal formation and the requirements of pedestal formation by host adapted and in vitro cultivated EHEC. Our data indicate that growing EHEC in the mammalian host enhances bacterial cell attachment, expression and translocation of virulence effectors and actin signaling, and this enhancement is likely to entail more than one bacterial activity involved in host cell interactions. We also focused on the interaction between the two key bacterial players involved in pedestal formation, intimin and Tir. We randomly mutagenized the Tir-binding domain of intimin and isolated point mutants that disrupted Tir recognition. The ability of intimin mutants to bind to recombinant Tir correlated with their ability to trigger AE lesions on pre-infected mammalian cells. Half of the mutations fell within the previously identified 50 amino acid C-terminal region of intimin, and alanine scanning mutagenesis of this region identified four residues of EHEC intimin that are critical for Tir recognition. In a model of the EHEC intimin-Tir complex that is based on EPEC intimin and Tir, these four amino acids are predicted to be located at the intimin-Tir interface, indicating that these residues play a functional role in intimin recognition by Tir. To identify critical residues involved in intimin recognition and intimin mediated actin signaling, we generated point mutations in the extracellular domain of EHEC Tir. Based on our data, we conclude that Tir-intimin interaction is essential for triggering actin pedestals, and intimin function in the context of Tir signaling can be replaced by proteins that are entirely unrelated to intimin but that bind to Tir. These data are concordant with the model that intimin functions to cluster Tir in the membrane to induce actin assembly. Finally, as a step to study downstream actin signaling processes after Tir translocation, we mapped the domain of Tir involved in host cell signaling. We found that the clustering of a 12 amino acid stretch of C-terminus encompassing the Nck binding sequence of Tir generated actin nucleation indistinguishable from that mediated by the entire C-terminus, and abrogation of Nck binding by mutation of Y474 to Phenylalanine abolished actin assembly. Although these results do not rule out a role for other domains of Tir involved in actin pedestal formation, this suggests that the essential element of Tir consists of the Nck binding domain.

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX. / Dissertation

Cells have evolved different pathways in order to tolerate damage produced by different cytotoxic agents. Each agent reacts differently with DNA causing formation of different types of adducts, each eliciting the SOS stress response to induce different cellular repair pathways. One such type of substrate generated by cytotoxic agents is the DNA double strand break (DSB). The main pathway to repair such damage in the cell is through a process of recombination. In this thesis, I specifically examined the anti-cancer therapeutic agent cisplatin, which forms single- and double-strand breaks in DNA, and methylating agents, which are proposed to also be capable of forming such breaks. Neither type of agent can directly form these breaks; however, they leave a signature type of damage lesion which is recognized by different repair processes. The mismatch repair (MMR) status of a mammalian cell or an Escherichia coli dam mutant relates directly to the sensitivity of the cells to the agents mentioned above. As the dam gene product plays an important role in this pathway and in other processes in the cell, when mutated, dam cells are more sensitive to methylating agents and cisplatin than wildtype. A combination of dam and either mutS or mutL restores resistance to the same agents to wild type levels. Therefore, mismatch repair sensitizes dam bacteria to these agents. The rationale for this comes from examining the viability of dam mutants, as dammutants are only viable because they are highly recombinogenic. The presence of MMR-induced nicks or gaps results in the formation of DSBs that require recombination to restore genomic integrity. Mismatch repair proteins inhibit recombination between homeologous DNA. Homeologous recombination (recombination between non-identical, but similar, DNA sequences) is only possible when the MMR proteins, MutS and MutL, are absent. It is postulated that this is because MutS recognizes the homeologous DNA and subsequently slows down or aborts recombination completely. The double mutant, dam mutS/L shows wild type levels of sensitivity to cisplatin because mismatch repair is no longer recognizing the adducts and recombinational repair is allowed to continue. Human cells behave in an analogous fashion to the bacterial dam mutant, showing sensitivity to cisplatin and methylating agents. When an additional mutation in a mismatch repair gene is present, the cells become as resistant as wild type. Therefore, the E. coli dammutant is a useful model system to study this mechanism of drug resistance. DNA containing cisplatin adducts or lesions resulting from methylation are substrates for other types of repair processes such as nucleotide excision repair and base excision repair; however they have also been implicated as substrates for MMR and recombinational repair. The goal of the work in this thesis was two-fold. The first was to identify the gene products and mechanism necessary for repair of cisplatin damage by recombination. The second was to examine the mechanism of cisplatin toxicity, and specifically how MMR proficiency aids in the cytotoxicity of this drug by preventing recombination. Using the duplicated inactive lac operon recombination assay, we were able to determine the requirements for spontaneous and cisplatin-induced recombination, the RecBCD and RecFOR pathways. We were also able to further postulate that the cisplatin- induced signature damage recognized by recombination was the double strand break, likely formed from fork stalling and regression or a subsequent collapse during DNA synthesis, thus requiring these pathways for repair. This observation led to the experiments involving examination of the mechanism of cisplatin toxicity and where MMR could inhibit specific steps of recombination with DNA containing cisplatin lesions. Low levels of cisplatin lesions slowed the rate of RecA-mediated strand transfer in vitro, likely due to its ability to form a large bend in the DNA. MutS bound to cisplatin lesions in the DNA during heteroduplex formation in the RecA strand exchange step of recombination, inhibiting branch migration, and aborting the reaction. In order for MutS to inhibit recombination with cisplatin lesions, the results in the work in Chapter IV, show that binding to the lesion requires the C-terminus of MutS to be present, possibly due to a requirement for tetramerization of the protein, a domain contained in the C-terminus of MutS. This antirecombination function is different than the mutation avoidance function of MutS, as binding of mismatches requires only dimers. This differential sensitivity for cisplatin versus a mismatch was further exemplified in Chapter V, the experiments with dna mutants, where the greatest difference in sensitivity was observed for a dnaE mutant (catalytic subunit of polIII), which was as sensitive to cisplatin as a dam mutant, but fairly resistant to treatment with MNNG. This is indicative of the potency of a cisplatin adduct to block polymerase progression, versus a mismatch which poses little problem to synthesis. Recombination is invoked to repair DSBs caused by the cisplatin lesions through the RecBCD and FOR pathways after fork regression or collapse. A main conclusion from these studies is that a cisplatin lesion is processed differently than a mismatch. The mechanism of how a cisplatin lesion is processed, forming the DSB which invokes recombinational repair is still unclear and continues to be investigated.

A steady state level of negative supercoiling is essential for chromosome condensation, initiation of replication and subsequent elongation step. DNA gyrase, found in every eubacteria, serves the essential housekeeping function of maintenance of the negative supercoiling status of the genome. The functional holoenzyme is a heterotetramer, comprising of two GyrA and two GyrB subunits. DNA gyrase is an indispensable enzyme and serves as a readily susceptible target for natural antibacterial agents. The enzymatic steps of topoisomerisation by gyrase involve transient double strand break and rejoining of the strands after intact duplex transfer. Corruption of its catalytic cycle can lead to the generation of cytotoxic double-strand DNA breaks. Most of the anti-gyrase agents achieve their objective by targeting the vulnerable step of the reaction cycle i.e. DNA cleavage step. Bacteria on their part must have evolved and adopted strategies to counter the action of external agents and prevent the generation of double strand breaks thereby safeguarding their genome. In the present thesis, attempts have been made to understand the role of three endogenous gyrase interacting proteins in gyrase modulation and cellular defense against anti-gyrase agents. The thesis is divided into six chapters. Chapter 1 introduces the wonder enzymes “DNA topoisomerases” starting with a brief classification of these enzymes and their physiological functions. In the next section, DNA gyrase has been discussed in greater detail. The structural aspects as well as the mechanism of the topoisomerisation reaction catalyzed by gyrase have been discussed. Final section gives an overview of different gyrase modulators known till date focusing on their source, structure and mode of action. The scope and objectives of the present study is presented at the end of this chapter. In Chapter 2 is aimed at understanding the physiological role of GyrI. GyrI, originally identified in Escherichia coli as an inhibitor of DNA gyrase, has been previously shown in the laboratory to render protection against gyrase poisons and also various other DNA damaging agents (mitomycin C, MNNG). Abolishing GyrI expression renders the cell hypersensitive to these cytotoxic agents. Interestingly, GyrI exhibits contrasting behavior towards two plasmid encoded proteinaceous poisons of DNA gyrase. It reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin. However, a positive cooperation between GyrI and F plasmid encoded toxin CcdB, results in enhanced DNA damage and cell death. These results suggest a more complex functional interplay and physiological role for GyrI. Search for other chromosomally encoded gyrase inhibitors led to YacG, a small zinc finger protein (7.3kDa) from E. coli, shown to be a member of DNA gyrase interactome, in a protein-protein interaction network described recently. Chapter 3 deals with the detailed characterization of YacG. It is shown that YacG inhibits DNA gyrase by binding to GyrB subunit and preventing DNA binding activity of the enzyme. More importantly, it protects against the cytotoxic effects of other gyrase inhibitors like ciprofloxacin, novobiocin, microcin B17 and CcdB. Further investigations revealed that YacG and its homologues are found only in proteobacteria. Hence, it appears to be a defense strategy developed by gram-negative bacteria to fight against the gyrase targeting cytotoxic agents. Inhibition by YacG appears to be specific to E. coli gyrase as mycobacterial enzyme is refractile to YacG action. GyrB, only in gram-negative organisms, possesses extra stretch of 165 amino acids, indispensable for DNA binding. Biochemical experiments with the truncated GyrB lacking the extra stretch reveal the importance of this stretch for stable YacG-GyrB interaction. E. coli topoisomerase IV is also resistant to YacG mediated inhibition, probably due to the absence of the extra stretch in ParE subunit, which is otherwise highly similar to GyrB. Further, YacG homologues from other proteobacterial members (Sinorhizobium meliloti and Haemophilus influenzae homologues sharing 35% and 63 % identity with E. coli YacG respectively ) also inhibits E. coli DNA gyrase at comparable levels. YacG thus emerges as a proteobacteria specific inhibitor of DNA gyrase. The occurrence of both YacG and the gyrase extra stretch only in proteobacteria, suggest co-evolution of interacting partners in proteobacteria. In Chapter 4, the study of endogenous gyrase modulators is extended to Mycobacterium sp. glutamate racemase (MurI) from E. coli has been shown earlier to be an inhibitor of DNA gyrase. However, nothing much was known about its mode of action. MurI is an important enzyme in the cell wall biosynthesis pathway, which catalyses the conversion of L-glutamate to D-glutamate, an integral component of the bacterial cell wall. In this chapter, it is demonstrated that M. tuberculosis MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species specific as E. coli gyrase is also inhibited. However, it is gyrase specific as topoisomerase I activity remains unaltered. The mechanism of inhibition by MurI has been elucidated for the first time and it is shown that MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. Chapter 5 is the extension of the studies on glutamate racemase into another species, i.e. Mycobacterium smegmatis. DNA gyrase inhibition seems to be an additional attribute of some of the glutamate racemases, but not all, as Glr isozyme from B. subtilis has no effect on gyrase activity in spite of sharing a high degree of similarity with the gyrase inhibitory glutamate racemases. It is shown that like the M. tuberculosis MurI, M. smegmatis enzyme is also a bifunctional enzyme. It inhibits DNA gyrase in addition to its racemization activity. Further, overexpression of the enzyme in M. smegmatis provides protection to the organism against fluoroquinolones. DNA gyrase inhibitory property thus appears to be a typical characteristic of these MurI and seems to have evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double strand breaks in the genome. In the above chapters, it is shown that besides its crucial role in cell wall biosynthesis, mycobacterial MurI moon lights as DNA gyrase inhibitor. That the two activities exhibited by M. tuberculosis MurI are unlinked and independent of each other is demonstrated in Chapter 6. Racemization function of MurI is not essential for its gyrase inhibitory property as mutants compromised in racemization activity retain gyrase inhibition property. MurI- DNA gyrase interaction influences gyrase activity but has no effect on racemization activity of MurI. MurI expression in mycobacterial cells provides protection against the action of ciprofloxacin, thereby suggesting a role of MurI in countering external agents targeting DNA gyrase. Further M. tuberculosis MurI overexpressed in near homologous expression system of M. smegmatis yields highly soluble enzyme which can be further used for structural and functional studies. In conclusion, the studies reveal that the endogenous inhibitors essentially influence the enzyme activity by sequestering the enzyme away from DNA. None of them cause cytotoxicity, which usually arises as a result of DNA damage caused by accumulation of gyrase-DNA covalent intermediate. On the contrary they provide protection against such gyrase poisons. Comparative analysis of these proteinaceous inhibitors, however, does not reveal a common motif or structural fold, required for their ability to inhibit DNA gyrase. Based on these studies, it can be proposed that these endogenous proteins exist to serve as cellular defense strategies against external abuse and also to modulate the intracellular activity of DNA gyrase as and when required, for accurate division, functioning and survival of the cells.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.