Bacterial Enteropathogen Research Articles

Mo1806 Helminth-Induced Alterations in the Gut Microbiota Exacerbate the Colitis Caused by Infection With a Bacterial Enteropathogen

Mo1806 Helminth-Induced Alterations in the Gut Microbiota Exacerbate the Colitis Caused by Infection With a Bacterial Enteropathogen

Diarrheagenic Escherichia coli carrying supplementary virulence genes are an important cause of moderate to severe diarrhoeal disease in Mexico.

Diarrheagenic Escherichia coli (DEC) cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG), mainly associated with enteroaggregative E. coli (EAEC), were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%), surpassing Salmonella (12%) and Shigella (9%). Predominant DEC groups were diffusely adherent E. coli (DAEC) (35%), EAEC (24%), and enteropathogenic E. coli (EPEC) (19%). Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26%) and DAEC (18%); 30% had moderate diarrhoea mainly caused by DAEC (36%), mixed DEC infections (33%) and EAEC (32%). DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6–24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3–13.9). The presence of SVG dispersin, (aatA), dispersin-translocator (aatA), enteroaggregative heat-stable toxin 1 (astA), plasmid encoded toxin (pet), cytolethal distending toxin (cdt) was higher in DEC than non-DEC strains, (36% vs 26%, P <0.0001, OR = 1.5, 95% CI, 1.3–1.8). 98% of EAEC-infected children harboured strains with SVG; 85% carried the aap-aatA gene combination, and 33% of these also carried astA. 28% of both EPEC and ETEC, and 6% of DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2%) and non-DEC strains (21% and 13%). DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children.

Thymol disrupts the membrane integrity of Salmonella ser. typhimurium in vitro and recovers infected macrophages from oxidative stress in an ex vivo model

Salmonella is a common bacterial enteropathogen responsible for many deaths every year. In the present study, we evaluated the mechanism of action of thymol against Salmonella ser. typhimurium, as well as its potential to induce intracellular killing and recovery from oxidative stress in macrophages. The minimum inhibitory concentration (MIC) of thymol against S. typhimurium was found to be 750 mg/l, and the CFU count decreased in a time-dependent manner. Excessive release of cellular materials and potassium ion also occurred in a time-dependent manner. Scanning electron microscopy showed disruption of membrane integrity. Intracellular killing capacity of macrophages was enhanced upon thymol treatment compared to control untreated cells. Thymol significantly reduced production of nitric oxide in a time-dependent manner, as well as the glutathione level. Disruption of membrane integrity was confirmed as the principle mechanism of action of thymol against S. typhimurium. Further, its potent role in inducing intracellular killing of S. typhimurium and recovery from oxidative stress in macrophages suggests that thymol can be applied as a naturally occurring drug against S. typhimurium in place of synthetic drugs.

Development of Fatal Intestinal Inflammation in MyD88 Deficient Mice Co-infected with Helminth and Bacterial Enteropathogens

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To determine whether and how an intestinal helminth parasite, Heligomosomoides polygyrus, might impact the TLR signaling pathway during the response to a bacterial enteropathogen, MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus, the bacterial enteropathogen Citrobacter rodentium, or both. We found that MyD88 knockout mice co-infected with H. polygyrus and C. rodentium developed more severe intestinal inflammation and elevated mortality compared to the wild-type mice. The enhanced susceptibility to C. rodentium, intestinal injury and mortality of the co-infected MyD88 knockout mice were found to be associated with markedly reduced intestinal phagocyte recruitment, decreased expression of the chemoattractant KC, and a significant increase in bacterial translocation. Moreover, the increase in bacterial infection and disease severity were found to be correlated with a significant downregulation of antimicrobial peptide expression in the intestinal tissue in co-infected MyD88 knockout mice. Our results suggest that the MyD88 signaling pathway plays a critical role for host defense and survival during helminth and enteric bacterial co-infection.

Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level.

Clinical study of acute childhood diarrhoea caused by bacterial enteropathogens.

There are not a large number of studies in India which can enlighten us regarding acute childhood diarrhoea and far lesser in number when it comes to its bacterial enteropathogenesis. The present study is specially targeted to determine the prevalence of various bacterial enteropathogens causing acute childhood diarrhoea and to find out their respective pattern of clinical features. All children under 12 years of age enrolled between 1st June, 2012 and 31st July 2012, in the Outpatient department, Inpatient department of pediatrics section and casualty of hospital, who presented with acute diarrhoea. Data collected by mean of study questionnaire. Stool sample were processed for bacteriological analysis. In 280 samples bacteria were isolated with the help of microscopy, culture and biochemical reactions. The isolates obtained were tested for antimicrobial sensitivity over Mueller Hinton agar by Kirby Bauer-disk diffusion method. Out of 280 children frequency of diarrhoeagenic bacteria isolated from the samples showed that Escherichia coli was recorded as the predominant bacteria with 44.2% of prevalence followed by Shigella, Salmonella, Klebsiella and Campylobacter with 28.2%, 13.6%, 7.8% and 6.1% respectively. Patients falling in the age group of 1-3 years. were the major sufferers of diarrhoea due to all etiologies except Klebsiella which mainly had impact on the patients below six months. Majority of isolated bacterial agents were resistant to Co-trimoxazole and Shigella being highly resistant enteropathogen isolated. Salmonella spp. were least resistant isolates. None of the isolates were resistant to Cefotaxime, Cefuroxime and Azetronam. RESULTS of study reveal that Escherichia coli is a predominant bacterial enteropathogen causing diarrhoea and Salmonella is a major contributor to the diarrhoea causing severe dehydration and to the clinical features like fever, vomiting and more than 10 times of frequency of stools. Shigella is among highly resistant isolates while Salmonella isolates had least resistance to majority of antibiotics.

Clay mineral type effect on bacterial enteropathogen survival in soil

Enteropathogens released into the environment can represent a serious risk to public health. Soil clay content has long been known to have an important effect on enteropathogen survival in soil, generally enhancing survival. However, clay mineral composition in soils varies, and different clay minerals have specific physiochemical properties that would be expected to impact differentially on survival. This work investigated the effect of clay materials, with a predominance of a particular mineral type (montmorillonite, kaolinite, or illite), on the survival in soil microcosms over 96days of Listeria monocytogenes, Salmonella Dublin, and Escherichia coli O157. Clay mineral addition was found to alter a number of physicochemical parameters in soil, including cation exchange capacity and surface area, and this was specific to the mineral type. Clay mineral addition enhanced enteropathogen survival in soil. The type of clay mineral was found to differentially affect enteropathogen survival and the effect was enteropathogen-specific.

A Mouse Model of Salmonella Typhi Infection

Salmonella spp. are gram-negative flagellated bacteria that can cause food- and waterborne gastroenteritis and typhoid fever in humans. We now report that flagellin from Salmonella spp. is recognized in mouse intestine by Toll-like receptor 11 (TLR11). Absence of TLR11 renders mice more susceptible to infection by S.Typhimurium, with increased dissemination of the bacteria and enhanced lethality. Unlike S.Typhimurium, S.Typhi, a human obligatory pathogen that causes typhoid fever, is normally unable to infect mice. TLR11 is expressed in mice, but not in humans, and remarkably, we find that tlr11(-/-) mice are efficiently infected with orally administered S.Typhi. We also find that tlr11(-/-) mice can be immunized against S.Typhi. Therefore, tlr11(-/-) mice represent a small-animal model for the study of the immune response to S.Typhi and for the development of vaccines against this important human pathogen.

Campylobacter— An extraordinary intestinal bacterium

This edition of the European Journal of Microbiology and Immunology is devoted to an extraordinary intestinal bacte­rium. Not only has Campylobacter outnumbered Salmonella as the most prevalent bacterial pathogen causing diarrhoea, but it also differs from other enteropathogenic bacteria in its epidemiology, its peculiar cell surface glycans, and its ab­sence of typical virulence factors, such as enterotoxins. Whereas infection of humans in developed countries is symptomatic irrespective of age in most cases, data from sub­Saharan Africa indicate that symptoms are mainly present in children, whereas frequent exposure might lead to asymp­tomatic colonization of adults. This raises questions such as whether differences in lifestyle, especially eating habits, more frequent exposure, and premunitive immunity in developing countries, or just strain variations are possible factors being involved. The identification of metabolic preferences and chemotaxis allowed the identification of new biological markers which might make source tracking become feasible in the future. However, it is still unclear as to why Campylobacter has become such a prevalent bacterial enteropathogen especially in developed countries. Is it because we eat more poultry, is it because the technique of slaughtering (poultry hang with the cloacae on top) might lead to fecal contamination of the body skin, or is it simply because diagnostic techniques have improved over time? The recent finding of coccoid forms of Campylobacter also points to the questions on how Campylobacter is responding to environmental stress factors and whether the trend of global warming has changed only our leisure behaviour or has it also increased the burden of Campylobacter in the environment. Equally important is the fact that Campylobacter can frequently be found in the intestine of its natural reservoir, such as poultry and other animals. Knowing that the optimal growth temperature for Campylobacter is above 40 °C makes it understandable that this bug prefers to colonize the avian intestine, since birds have such a high body temperature. This, however, raises another interesting question: do different strains circulate in birds, mammals, and the environment, and does the temperature regulate the expression of virulence-associated factors, and if so, which bacterial factors have to be focused on? In this regard, the development of innovative murine animal models will facilitate our understanding of the patho­genesis of campylobacteriosis. It has recently been shown that the composition of the gut flora as well as the composition of food made colonization-resistant mice become susceptible to Campylobacter infection. These in vivo models provide a great chance to significantly improve our understanding on what determines virulence of Campylobacter. Some of the most important European scientists working on Campylobacter came together in October 2011 at the CampyGerm’s Campylobacter Conference * in Gottingen/Germany to discuss these and other questions. Some of the presented data are published here. It is hoped that this extraordinary bacterium will attract more scientists to decipher its multiple secrets and to open up new doors that finally will allow better prevention, control, and management of Campy­lobacter infection of man and animals. Prof. Dr. Uwe Gros, Department of Microbiology and Immunology, University of Gottingen, Germany

A sorting platform determines the order of protein secretion in bacterial type III systems.

Bacterial type III protein secretion systems deliver effector proteins into eukaryotic cells in order to modulate cellular processes. Central to the function of these protein-delivery machines is their ability to recognize and secrete substrates in a defined order. Here, we describe a mechanism by which a type III secretion system from the bacterial enteropathogen Salmonella enterica serovar Typhimurium can sort its substrates before secretion. This mechanism involves a cytoplasmic sorting platform that is sequentially loaded with the appropriate secreted proteins. The sequential loading of this platform, facilitated by customized chaperones, ensures the hierarchy in type III protein secretion. Given the presence of these machines in many important pathogens, these findings can serve as the bases for the development of novel antimicrobial strategies.

Rotavirus Disease

Background: Many rotavirus infections occur as mixed infections with other microorganisms, including viruses, bacteria, and protozoa. In vitro, animal and clinical studies provide evidence for potentiation between rotavirus infection and coinfecting pathogens. The mechanisms of such interactions are not well studied or understood. Methods: A literature search was performed to identify publications on rotavirus coinfections. Results: The evidence for potentiation between infections with rotavirus and bacterial enteropathogens—particularly E. coli—is strong. A number of large clinical investigations has shown that children infected with rotavirus and a bacterial enteropathogen suffer from more severe diarrhea and/or dehydration, with diarrhea often lasting longer than in children infected with rotavirus alone. In general, but not exclusively, coinfections result in a poorer prognosis. Conclusions: In developing countries, rotavirus vaccination could possibly improve the poor prognosis associated with bacterial enteropathogen-rotavirus coinfections. However, further studies are needed to define the exact role of rotavirus vaccination in the context of coinfections.

Fate of bacteria, Aeromonas caviae, in the midgut of the housefly, Musca domestica

Abstract. The role of houseflies as agents in the spread of bacterial diseases has been thoroughly investigated, yet the fate of bacteria ingested by flies has not. We examined the physical location of the bacterial enteropathogen Aeromonas caviae in the midgut of laboratory‐reared adult houseflies. Food ingested by houseflies was separated from the midgut epithelium by a double‐layered peritrophic matrix (PM). The inner PM intimately enveloped the food as fecal pellets (food boluses), while the outer PM appeared as a long continuous tube. In flies fed a suspension of A. caviae, live bacteria were not observed within the inner PM, but were compartmentalized between folds of the PM in the inter‐PM space. Similar observations were made for flies fed a suspension of Serratia liquefaciens and for highly contaminated feral flies. Isolates of both A. caviae and S. liquefaciens were chitinolytic (as demonstrated by clearing zones on chitin agar), but the potential role of bacterial enzymes in the alteration of PM morphology or formation needs further investigation.

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.

Enterohemorrhagic Escherichia coli O157:H7 disrupts Stat1-mediated gamma interferon signal transduction in epithelial cells.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a clinically important bacterial enteropathogen that manipulates a variety of host cell signal transduction cascades to establish infection. However, the effect of EHEC O157:H7 on Jak/Stat signaling is unknown. To define the effect of EHEC infection on epithelial gamma interferon (IFN-gamma)-Stat1 signaling, human T84 and HEp-2 epithelial cells were infected with EHEC O157:H7 and then stimulated with recombinant human IFN-gamma. Cells were also infected with different EHEC strains, heat-killed EHEC, enteropathogenic E. coli (EPEC) O127:H6, and the commensal strain E. coli HB101. Nuclear and whole-cell protein extracts were prepared and were assayed by an electrophoretic mobility shift assay (EMSA) and by Western blotting, respectively. Cells were also processed for immunofluorescence to detect the subcellular localization of Stat1. The EMSA revealed inducible, but not constitutive, Stat1 activation upon IFN-gamma treatment of both cell lines. The EMSA also showed that 6 h of EHEC O157:H7 infection, but not 30 min of EHEC O157:H7 infection, prevented subsequent Stat1 DNA binding induced by IFN-gamma, whereas infection with EPEC did not. Immunoblotting showed that infection with EHEC, but not infection with EPEC, eliminated IFN-gamma-induced Stat1 tyrosine phosphorylation in both dose- and time-dependent fashions and disrupted inducible protein expression of the Stat1-dependent gene interferon regulatory factor 1. Immunofluorescence revealed that EHEC infection did not prevent nuclear accumulation of Stat1 after IFN-gamma treatment. Also, Stat1 tyrosine phosphorylation was suppressed by different EHEC isolates, including intimin-, type III secretion- and plasmid-deficient strains, but not by HB101 and heat-killed EHEC. These findings indicate the novel disruption of host cell signaling caused by EHEC infection but not by EPEC infection.

Shiga toxin-producing E coli : Two tests are better than one

Shiga toxin-producing E coli : Two tests are better than one

Generation of Campylobacter jejuni genetic diversity in vivo.

Molecular epidemiology studies suggest that horizontal genetic exchange is a major cause of pathogen biodiversity. We tested this concept for the bacterial enteropathogen Campylobacter jejuni by seeking direct in vivo evidence for the exchange of genetic material among Campylobacter strains. For this purpose, two antibiotic resistance markers were inserted into the hipO or htrA gene of genetically distinct and naturally transformable C. jejuni strains. Genetic exchange of the resistance markers was analysed after co-cultivation of homologous and heterologous strains in vitro and in vivo during experimental infection of chickens. Double-resistant recombinants were obtained both in vitro and from the chicken intestine for all combinations of strains tested. Bidirectional genetic exchange of DNA between homologous and heterologous strains was confirmed by Southern blotting in combination with flaA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE). Extensive PFGE analyses of isolated recombinants indicated the frequent occurrence of genetic rearrangements during the experimental infection, in addition to the homologous recombination of the antibiotic resistance genes. Together, the data indicate unequivocally that interstrain genetic exchange as well as intragenomic alterations do occur in vivo during C. jejuni infection. These events probably explain the genome plasticity observed for this pathogen.

Incidence of Clostridium perfringens in Broiler Chickens and Their Environment during Production and Processing

During a calendar year, a study was conducted involving 16 broiler flocks on four different farms, two farms belonging to each of two major U.S. poultry integrators. As determined by the detection of Clostridium perfringens in fecal or cecal samples, 15 (94%) of the flocks became positive for this bacterial enteropathogen, and only one remained negative throughout the 6-to-8-wk rearing period. Paper pads beneath chicks that were transported from the hatchery to the rearing house were contaminated with C. perfringens in 15 (94%) of the flocks. When sampled biweekly through grow out, 13 of the flocks were C. perfringens positive at 2 wk of age. These results suggest that colonization of the intestinal tract of broilers by C. perfringens is an early event. Of the environmental samples, all but feed in the hopper were contaminated before placement for at least one of the rearing periods. All sample types were contaminated at some point during the 6-to-8-wk grow-out period. Of the on-farm environmental samples, the highest incidences (percentage positive) of C. perfringens were detected in wall swabs (53%), fan swabs (46%), fly strips (43%), dirt outside the house entrance (43%), and swabs of workers' boots (29%). Birds were usually transported to the processing plant in coops that were already contaminated with C. perfringens. In the plant, C. perfringens was isolated more frequently from samples of scald water than from those of chill water. Clostridium perfringens was recovered from broiler carcasses after chilling in 13 (81%) of the 16 flocks. The proportion of C. perfringens-positive carcasses for the contaminated flocks ranged from 8% to 68% with a mean of 30%.

SopE and SopE2 from Salmonella typhimurium Activate Different Sets of RhoGTPases of the Host Cell

The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but not with Rac1. Affinity precipitation of Cdc42.GTP and Rac1.GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allow S. typhimurium to specifically activate different sets of RhoGTPase signaling cascades.

Escherichia coli infections and malnutrition

In a worldwide study of morbidity and mortality based on population estimates in 1980 it was found that 744 million to 1 billion episodes of diarrhea and 4.6 million diarrheal deaths were recorded each year in that decade in children younger than 5 years in Africa Asia and Latin America. A decade later despite improvements in management which reduced worldwide mortality to 3.3 million deaths per year the incidence of diarrhea remained the same. Acute diarrhea is still the most important symptom associated with malnutrition. In Brazil infants younger than 1 year of age represent the age group with a higher than average risk of death due to diarrhea. Studies have found that enteropathogenic Escherichia coli (EPEC) was the most frequent bacterial enteropathogen identified in stool samples of these infants. In general EPEC infections are more commonly associated with severe acute diarrhea and can potentially evolve to a protracted illness leading to food intolerance and malnutrition in up to 30% of cases. In the small intestinal surface of 16 infants with persistent diarrhea associated with severe malnutrition and serious food intolerance EPEC was isolated from stool samples. An aggressive therapeutic approach with appropriate nutritional support and the adequate correction of fecal losses are required to allow complete recovery from the damage caused by this enteropathogenic agent.

Purification and biochemical activity of Salmonella exchange factor SopE

This chapter discusses the purification and biochemical activity of salmonella typhimurium exchange factor SopE. Salmonella typhimurium is a bacterial enteropathogen responsible for a large percentage of bacterial food poisoning. SopE is one of the S. typhimurium effector proteins injected into host cells. SopE are thought to provide the signals necessary for recognition and injection into host cells by the type III secretion system. The chapter presents a detailed description of the preparation and of several biochemical assays to analyze the interaction of SopE with Rho guanosine 5'-triphosphate (GTPase).The chapter explains the process of overproduction and purification of recombinant SopE proteins. Preparation of Rho guanosine 5'-triphosphate GTPase proteins is also explained. The chapter discusses the filter-binding assay for SopE-mediated guanine nucleotide exchange. In the presence of native SopE , guanine nucleotide exchange rates of [ 3 H]GDP-loaded Cdc42 are enhanced greatly. The chapter presents surface plasmon resonance assay to study SopE- Cdc42 complex. The chapter concludes with a discussion on the specificity of SopE.