Stephen Calderwood - Academia.edu (original) (raw)

Papers by Stephen Calderwood

American Journal of Medicine, 1987

Using a large cohort of patients from the International Collaboration on Endocarditis Merged Data... more Using a large cohort of patients from the International Collaboration on Endocarditis Merged Database, we compared coagulase-negative staphylococcal (CoNS) native-valve endocarditis (NVE) to NVE caused by more common pathogens. Rates of heart failure and mortality were similar between patients with CoNS NVE and patients with Staphylococcus aureus NVE, but rates for both groups were significantly higher than rates for patients with NVE due to viridans streptococci. These results emphasize the importance of CoNS as a cause of NVE and the potential for serious complications with this infection. Coagulase-negative staphylococci (CoNS) accounts for ∼5% of all episodes of native-valve endocarditis (NVE) [1-3]. Rates of CoNS bacteremia [4, 5] and CoNS endocarditis [6] have increased, underscoring the need to improve our knowledge of this pathogen. Although prior case reports and retrospective series [6-9] have suggested high rates of complications among patients with NVE due to CoNS, our understanding of this

Biochemistry, 1992

Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eukaryotic ribosomes by catal... more Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eukaryotic ribosomes by catalytically depurinating adenosine 4324 in 28S rRNA. Although the primary structure of the enzymatic portion of the molecule (Slt-IA) is known to contain regions of significant homology to the ricin A chain (RTA), and although certain residues have been implicated in catalysis, the crystal structure of Slt-IA has not been solved nor has the geometry of its active site been well defined. In order to derive a more complete understanding of the nature of the Slt-IA active site, we placed the slt-IA gene under control of an inducible promoter in Saccharomyces cerevisiae. Induction of the cloned element was lethal to the host. This lethality was the basis for selection of an attenuated mutant of Slt-IA changed at tyrosine 77, a locus not previously linked to the active site. As well, it permitted evaluation of the toxicity of a number of mutant Slt-IA cassettes that we constructed in vitro. Putative active-site residues implicated in this fashion and in other studies were mapped to an energy-minimized computer model of Slt-IA that had been generated on the basis of the known crystal structure of RTA. A cleft was identified on one face of the protein in which all implicated residues clustered, irrespective of their distances from one another in the primary structure of the molecule. Many of the chemical features anticipated in the active site of an RNA N-glycosidase are indeed present on the amino acid side chains occupying the cleft.

PLOS Neglected Tropical Diseases, 2015

Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of ep... more Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of epidemic cholera reported globally is El Tor biotype Vibrio cholerae isolates of the serogroup O1. However, in Bangladesh, outbreaks attributed to V. cholerae serogroup O139 isolates, which fall within the same phylogenetic lineage as the O1 serogroup isolates, were seen between 1992 and 1993 and in 2002 to 2005. Since then, V. cholerae serogroup O139 has only been sporadically isolated in Bangladesh and is now rarely isolated elsewhere. Here, we present case histories of four cholera patients infected with V. cholerae serogroup O139 in 2013 and 2014 in Bangladesh. We comprehensively typed these isolates using conventional approaches, as well as by whole genome sequencing. Phenotypic typing and PCR confirmed all four isolates belonging to the O139 serogroup. Whole genome sequencing revealed that three of the isolates were phylogenetically closely related to previously sequenced El Tor biotype, pandemic 7, toxigenic V. cholerae O139 isolates originating from Bangladesh and elsewhere. The fourth isolate was a non-toxigenic V. cholerae that, by conventional approaches, typed as O139 serogroup but was genetically divergent from previously sequenced pandemic 7 V. cholerae lineages belonging to the O139 or O1 serogroups. These results suggest that previously observed lineages of V. cholerae O139 persist in Bangladesh and can cause clinical disease and that a novel disease-causing non-toxigenic O139 isolate also occurs.

Clinical and Vaccine Immunology, 2015

Vibrio choleraeand enterotoxigenicEscherichia coli(ETEC) are noninvasive mucosal pathogens that c... more Vibrio choleraeand enterotoxigenicEscherichia coli(ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates β7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. β7 is a cell surface marker associated with mucosal homing. We isolated β7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a ...

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, Jan 23, 2015

In 2001, a hybrid strain of Vibrio cholerae O1 El Tor that expresses a classical cholera toxin (C... more In 2001, a hybrid strain of Vibrio cholerae O1 El Tor that expresses a classical cholera toxin (CT) emerged and this hybrid variant rapidly replaced the previous El Tor strain around the world. The global emergence of this variant coincided with anecdotal reports that cholera patients were presenting with more severe dehydration and disease in many locations. We compared severity of disease in cholera patients from before and after emergence of the hybrid strain at a diarrheal hospital in Dhaka, Bangladesh. We did indeed find that cholera patients presented with more severe dehydration and severe disease in the latter period; however, this was also true for "all non-cholera patients" as well. In addition, in sub-analyses of patients who presented with rotavirus and enterotoxigenic E. coli (ETEC), we found similar results. Comparing the two periods for differences in patient characteristics, nutritional status, vaccination status and income, we were unable to detect a plaus...

Background: Characterizing the dynamic physiological state of a bacterial pathogen during the cou... more Background: Characterizing the dynamic physiological state of a bacterial pathogen during the course of human infection is technically challenging. Most studies have consequently focused on the use of in vitro conditions or animal models to define genetic programs important to virulence. Although the use of such models of Vibrio cholerae infection has led to advances in the understanding of cholera pathogenesis and microbial virulence, the correlation between genetic regulation in these models and the events occurring during natural human infection remains unclear. Methods: We applied microarray transcriptional profiling directly to V. cholerae during the early and late stages of human infection. V. cholerae from human vomitus represented the organism’s physiologic state in the human upper gastrointestinal tract, while V. cholerae from human stool represented the late phase of human infection. Results: Our results demonstrate that early human infection is associated with high levels...

Iron is an essential nutrient to support the growth of most bacterial species. However, iron is n... more Iron is an essential nutrient to support the growth of most bacterial species. However, iron is not easily available to microorganisms infecting mammalian hosts, because it is largely sequestered by iron-binding proteins, such as transferrin or lactoferrin, or complexed to heme. In response to environmental iron stress, Vibrio cholerae produces the siderophore vibriobactin as well as a number of iron-induced

We constructed a synthetic oligonucleotide corresponding to the previously proposed consensus bin... more We constructed a synthetic oligonucleotide corresponding to the previously proposed consensus binding site for the Fur protein, a central iron-regulatory protein of Escherichia coli. When this oligonucleotide was introduced at the start of transcription of an operon fusion between the ompF promoter and the lacZ structural gene, 3-galactosidase activity became iron regulated. This consensus sequence is sufficient to function as an operator site for the binding of Fur protein in vivo.

Representational difference analysis (RDA) is a recently developed technique used for amplifying ... more Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two Vibrio cholerae serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the O1 serogroup. With both techniques, we recovered several

Proceedings of the National Academy of Sciences, 2015

Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the... more Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA+ ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence...

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, Jan 14, 2015

The objective of this study was to examine the clinical and immunological features of cholera in ... more The objective of this study was to examine the clinical and immunological features of cholera in pregnancy. Women of reproductive age presenting to the icddr,b Dhaka hospital with cholera, and enrolled as part of a larger cohort study, were tested for pregnancy on admission. We compared initial clinical features and immune responses of pregnant patients with non-pregnant female patients at days 2, 7 and 21 after infection. Among reproductive age women enrolled between January 2001 and May 2006, 9.7% (14/144) were pregnant. The duration of diarrhea prior to admission tended to be higher in pregnant compared to non-pregnant patients (p=0.08), but other clinical characteristics did not differ. Antibody responses to cholera toxin B subunit (CtxB), toxin-coregulated pilus A (TcpA), Vibrio cholerae lipopolysaccharide (LPS), and serum vibriocidal antibody responses, were comparable between pregnant and non-pregnant patients. There were no deaths among the pregnant cases or non-pregnant con...

Bacterial strains and plasmids are listed in Table S1. V. cholerae AC53 was used as a wildtype st... more Bacterial strains and plasmids are listed in Table S1. V. cholerae AC53 was used as a wildtype strain. For genetic manipulations E. coli strains DH5α, DH5αλpir and SM10λpir were used. Unless stated otherwise, bacteria were grown in LB at 37°C with aeration. M9 was prepared using DifcoTM M9 Minimal Salts according to the instructions. Supplements were used in the following

PLoS neglected tropical diseases, 2015

Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is ... more Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization. Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In ge...

The American journal of tropical medicine and hygiene, Jan 8, 2015

Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infec... more Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infected humans. Leptin is a hormone that plays a role in both metabolism and mediating immune responses. We characterized leptin levels in 11 children with cholera in Bangladesh, assessing leptin levels on days 2, 7, 30, and 180 following cholera. We found that patients at the acute stage of cholera had significantly lower plasma leptin levels than matched controls, and compared with levels in late convalescence. We then assessed immune responses to V. cholerae antigens in 74 children with cholera, correlating these responses to plasma leptin levels on day 2 of illness. In multivariate analysis, we found an association between day 2 leptin levels and development of later anti-cholera toxin B subunit (CtxB) responses. This finding appeared to be limited to children with better nutritional status. Interestingly, we found no association between leptin levels and antibody responses to V. cholera...

Cryptococcal infections are a global cause of significant morbidity and mortality. We have shown ... more Cryptococcal infections are a global cause of significant morbidity and mortality. We have shown that C. neoformans kills the nematode C. elegans and that several C. neoformans genes previously shown to be involved in mammalian virulence also play a role in C. elegans killing (PNAS 99:15675, 2002). We are using the C. neoformans/C. elegans model to identify novel genes that are associated with evolutionarily conserved aspects of cryptococcal virulence. Methods: We used the C. elegans/C. neoformans model to screen a bank of random insertional mutants that were developed in the C. neoformans H99 background. DNA flanking insertions in mutants with attenuated virulence was obtained by inverse PCR. Mutants are crossed to a pathogenic wild type strain to confirm linkage between the insertions and attenuated virulence phenotypes. Results: Among 300 mutants of C. neoformans that we have screened so far, we found that 15 (5%) of these mutants were attenuated in the initial C. elegans screen ...

PLoS neglected tropical diseases, 2015

Children bear a large burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Ty... more Children bear a large burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Typhi) in endemic areas. However, immune responses and clinical findings in children are not well defined. Here, we describe clinical and immunological characteristics of young children with S. Typhi bacteremia, and antimicrobial susceptibility patterns of isolated strains. As a marker of recent infection, we have previously characterized antibody-in-lymphocyte secretion (TPTest) during acute typhoid fever in adults. We similarly assessed membrane preparation (MP) IgA responses in young children at clinical presentation, and then 7-10 days and 21-28 days later. We also assessed plasma IgA, IgG and IgM responses and T cell proliferation responses to MP at these time points. We compared responses in young children (1-5 years) with those seen in older children (6-17 years), adults (18-59 years), and age-matched healthy controls. We found that, compared to age-matched controls patients in all ...

Infection and immunity, 1999

Epidemic strains of Vibrio cholerae O1 are divided into two biotypes, classical and El Tor. In bo... more Epidemic strains of Vibrio cholerae O1 are divided into two biotypes, classical and El Tor. In both biotypes, regulation of virulence gene expression depends on a cascade in which ToxR activates expression of ToxT, and ToxT activates expression of cholera toxin and other virulence genes. In the classical biotype, maximal expression of this ToxR regulon in vitro occurs at 30 degrees C at pH 6.5 (ToxR-inducing conditions), whereas in the El Tor biotype, production of these virulence genes only occurs under very limited conditions and not in response to temperature and pH; this difference between biotypes is mediated at the level of toxT transcription. In the classical biotype, two other proteins, TcpP and TcpH, are needed for maximal toxT transcription. Transcription of tcpPH in the classical biotype is regulated by pH and temperature independently of ToxR or ToxT, suggesting that TcpP and TcpH couple environmental signals to transcription of toxT. In this study, we show a near absenc...

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, Jan 24, 2015

The Journal of infectious diseases, 2015

American Journal of Medicine, 1987

Using a large cohort of patients from the International Collaboration on Endocarditis Merged Data... more Using a large cohort of patients from the International Collaboration on Endocarditis Merged Database, we compared coagulase-negative staphylococcal (CoNS) native-valve endocarditis (NVE) to NVE caused by more common pathogens. Rates of heart failure and mortality were similar between patients with CoNS NVE and patients with Staphylococcus aureus NVE, but rates for both groups were significantly higher than rates for patients with NVE due to viridans streptococci. These results emphasize the importance of CoNS as a cause of NVE and the potential for serious complications with this infection. Coagulase-negative staphylococci (CoNS) accounts for ∼5% of all episodes of native-valve endocarditis (NVE) [1-3]. Rates of CoNS bacteremia [4, 5] and CoNS endocarditis [6] have increased, underscoring the need to improve our knowledge of this pathogen. Although prior case reports and retrospective series [6-9] have suggested high rates of complications among patients with NVE due to CoNS, our understanding of this

Biochemistry, 1992

Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eukaryotic ribosomes by catal... more Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eukaryotic ribosomes by catalytically depurinating adenosine 4324 in 28S rRNA. Although the primary structure of the enzymatic portion of the molecule (Slt-IA) is known to contain regions of significant homology to the ricin A chain (RTA), and although certain residues have been implicated in catalysis, the crystal structure of Slt-IA has not been solved nor has the geometry of its active site been well defined. In order to derive a more complete understanding of the nature of the Slt-IA active site, we placed the slt-IA gene under control of an inducible promoter in Saccharomyces cerevisiae. Induction of the cloned element was lethal to the host. This lethality was the basis for selection of an attenuated mutant of Slt-IA changed at tyrosine 77, a locus not previously linked to the active site. As well, it permitted evaluation of the toxicity of a number of mutant Slt-IA cassettes that we constructed in vitro. Putative active-site residues implicated in this fashion and in other studies were mapped to an energy-minimized computer model of Slt-IA that had been generated on the basis of the known crystal structure of RTA. A cleft was identified on one face of the protein in which all implicated residues clustered, irrespective of their distances from one another in the primary structure of the molecule. Many of the chemical features anticipated in the active site of an RNA N-glycosidase are indeed present on the amino acid side chains occupying the cleft.

PLOS Neglected Tropical Diseases, 2015

Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of ep... more Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of epidemic cholera reported globally is El Tor biotype Vibrio cholerae isolates of the serogroup O1. However, in Bangladesh, outbreaks attributed to V. cholerae serogroup O139 isolates, which fall within the same phylogenetic lineage as the O1 serogroup isolates, were seen between 1992 and 1993 and in 2002 to 2005. Since then, V. cholerae serogroup O139 has only been sporadically isolated in Bangladesh and is now rarely isolated elsewhere. Here, we present case histories of four cholera patients infected with V. cholerae serogroup O139 in 2013 and 2014 in Bangladesh. We comprehensively typed these isolates using conventional approaches, as well as by whole genome sequencing. Phenotypic typing and PCR confirmed all four isolates belonging to the O139 serogroup. Whole genome sequencing revealed that three of the isolates were phylogenetically closely related to previously sequenced El Tor biotype, pandemic 7, toxigenic V. cholerae O139 isolates originating from Bangladesh and elsewhere. The fourth isolate was a non-toxigenic V. cholerae that, by conventional approaches, typed as O139 serogroup but was genetically divergent from previously sequenced pandemic 7 V. cholerae lineages belonging to the O139 or O1 serogroups. These results suggest that previously observed lineages of V. cholerae O139 persist in Bangladesh and can cause clinical disease and that a novel disease-causing non-toxigenic O139 isolate also occurs.

Clinical and Vaccine Immunology, 2015

Vibrio choleraeand enterotoxigenicEscherichia coli(ETEC) are noninvasive mucosal pathogens that c... more Vibrio choleraeand enterotoxigenicEscherichia coli(ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates β7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. β7 is a cell surface marker associated with mucosal homing. We isolated β7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a ...

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, Jan 23, 2015

In 2001, a hybrid strain of Vibrio cholerae O1 El Tor that expresses a classical cholera toxin (C... more In 2001, a hybrid strain of Vibrio cholerae O1 El Tor that expresses a classical cholera toxin (CT) emerged and this hybrid variant rapidly replaced the previous El Tor strain around the world. The global emergence of this variant coincided with anecdotal reports that cholera patients were presenting with more severe dehydration and disease in many locations. We compared severity of disease in cholera patients from before and after emergence of the hybrid strain at a diarrheal hospital in Dhaka, Bangladesh. We did indeed find that cholera patients presented with more severe dehydration and severe disease in the latter period; however, this was also true for "all non-cholera patients" as well. In addition, in sub-analyses of patients who presented with rotavirus and enterotoxigenic E. coli (ETEC), we found similar results. Comparing the two periods for differences in patient characteristics, nutritional status, vaccination status and income, we were unable to detect a plaus...

Background: Characterizing the dynamic physiological state of a bacterial pathogen during the cou... more Background: Characterizing the dynamic physiological state of a bacterial pathogen during the course of human infection is technically challenging. Most studies have consequently focused on the use of in vitro conditions or animal models to define genetic programs important to virulence. Although the use of such models of Vibrio cholerae infection has led to advances in the understanding of cholera pathogenesis and microbial virulence, the correlation between genetic regulation in these models and the events occurring during natural human infection remains unclear. Methods: We applied microarray transcriptional profiling directly to V. cholerae during the early and late stages of human infection. V. cholerae from human vomitus represented the organism’s physiologic state in the human upper gastrointestinal tract, while V. cholerae from human stool represented the late phase of human infection. Results: Our results demonstrate that early human infection is associated with high levels...

Iron is an essential nutrient to support the growth of most bacterial species. However, iron is n... more Iron is an essential nutrient to support the growth of most bacterial species. However, iron is not easily available to microorganisms infecting mammalian hosts, because it is largely sequestered by iron-binding proteins, such as transferrin or lactoferrin, or complexed to heme. In response to environmental iron stress, Vibrio cholerae produces the siderophore vibriobactin as well as a number of iron-induced

We constructed a synthetic oligonucleotide corresponding to the previously proposed consensus bin... more We constructed a synthetic oligonucleotide corresponding to the previously proposed consensus binding site for the Fur protein, a central iron-regulatory protein of Escherichia coli. When this oligonucleotide was introduced at the start of transcription of an operon fusion between the ompF promoter and the lacZ structural gene, 3-galactosidase activity became iron regulated. This consensus sequence is sufficient to function as an operator site for the binding of Fur protein in vivo.

Representational difference analysis (RDA) is a recently developed technique used for amplifying ... more Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two Vibrio cholerae serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the O1 serogroup. With both techniques, we recovered several

Proceedings of the National Academy of Sciences, 2015

Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the... more Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA+ ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence...

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, Jan 14, 2015

The objective of this study was to examine the clinical and immunological features of cholera in ... more The objective of this study was to examine the clinical and immunological features of cholera in pregnancy. Women of reproductive age presenting to the icddr,b Dhaka hospital with cholera, and enrolled as part of a larger cohort study, were tested for pregnancy on admission. We compared initial clinical features and immune responses of pregnant patients with non-pregnant female patients at days 2, 7 and 21 after infection. Among reproductive age women enrolled between January 2001 and May 2006, 9.7% (14/144) were pregnant. The duration of diarrhea prior to admission tended to be higher in pregnant compared to non-pregnant patients (p=0.08), but other clinical characteristics did not differ. Antibody responses to cholera toxin B subunit (CtxB), toxin-coregulated pilus A (TcpA), Vibrio cholerae lipopolysaccharide (LPS), and serum vibriocidal antibody responses, were comparable between pregnant and non-pregnant patients. There were no deaths among the pregnant cases or non-pregnant con...

Bacterial strains and plasmids are listed in Table S1. V. cholerae AC53 was used as a wildtype st... more Bacterial strains and plasmids are listed in Table S1. V. cholerae AC53 was used as a wildtype strain. For genetic manipulations E. coli strains DH5α, DH5αλpir and SM10λpir were used. Unless stated otherwise, bacteria were grown in LB at 37°C with aeration. M9 was prepared using DifcoTM M9 Minimal Salts according to the instructions. Supplements were used in the following

PLoS neglected tropical diseases, 2015

Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is ... more Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization. Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In ge...

The American journal of tropical medicine and hygiene, Jan 8, 2015

Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infec... more Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infected humans. Leptin is a hormone that plays a role in both metabolism and mediating immune responses. We characterized leptin levels in 11 children with cholera in Bangladesh, assessing leptin levels on days 2, 7, 30, and 180 following cholera. We found that patients at the acute stage of cholera had significantly lower plasma leptin levels than matched controls, and compared with levels in late convalescence. We then assessed immune responses to V. cholerae antigens in 74 children with cholera, correlating these responses to plasma leptin levels on day 2 of illness. In multivariate analysis, we found an association between day 2 leptin levels and development of later anti-cholera toxin B subunit (CtxB) responses. This finding appeared to be limited to children with better nutritional status. Interestingly, we found no association between leptin levels and antibody responses to V. cholera...

Cryptococcal infections are a global cause of significant morbidity and mortality. We have shown ... more Cryptococcal infections are a global cause of significant morbidity and mortality. We have shown that C. neoformans kills the nematode C. elegans and that several C. neoformans genes previously shown to be involved in mammalian virulence also play a role in C. elegans killing (PNAS 99:15675, 2002). We are using the C. neoformans/C. elegans model to identify novel genes that are associated with evolutionarily conserved aspects of cryptococcal virulence. Methods: We used the C. elegans/C. neoformans model to screen a bank of random insertional mutants that were developed in the C. neoformans H99 background. DNA flanking insertions in mutants with attenuated virulence was obtained by inverse PCR. Mutants are crossed to a pathogenic wild type strain to confirm linkage between the insertions and attenuated virulence phenotypes. Results: Among 300 mutants of C. neoformans that we have screened so far, we found that 15 (5%) of these mutants were attenuated in the initial C. elegans screen ...

PLoS neglected tropical diseases, 2015

Children bear a large burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Ty... more Children bear a large burden of typhoid fever caused by Salmonella enterica serotype Typhi (S. Typhi) in endemic areas. However, immune responses and clinical findings in children are not well defined. Here, we describe clinical and immunological characteristics of young children with S. Typhi bacteremia, and antimicrobial susceptibility patterns of isolated strains. As a marker of recent infection, we have previously characterized antibody-in-lymphocyte secretion (TPTest) during acute typhoid fever in adults. We similarly assessed membrane preparation (MP) IgA responses in young children at clinical presentation, and then 7-10 days and 21-28 days later. We also assessed plasma IgA, IgG and IgM responses and T cell proliferation responses to MP at these time points. We compared responses in young children (1-5 years) with those seen in older children (6-17 years), adults (18-59 years), and age-matched healthy controls. We found that, compared to age-matched controls patients in all ...

Infection and immunity, 1999

Epidemic strains of Vibrio cholerae O1 are divided into two biotypes, classical and El Tor. In bo... more Epidemic strains of Vibrio cholerae O1 are divided into two biotypes, classical and El Tor. In both biotypes, regulation of virulence gene expression depends on a cascade in which ToxR activates expression of ToxT, and ToxT activates expression of cholera toxin and other virulence genes. In the classical biotype, maximal expression of this ToxR regulon in vitro occurs at 30 degrees C at pH 6.5 (ToxR-inducing conditions), whereas in the El Tor biotype, production of these virulence genes only occurs under very limited conditions and not in response to temperature and pH; this difference between biotypes is mediated at the level of toxT transcription. In the classical biotype, two other proteins, TcpP and TcpH, are needed for maximal toxT transcription. Transcription of tcpPH in the classical biotype is regulated by pH and temperature independently of ToxR or ToxT, suggesting that TcpP and TcpH couple environmental signals to transcription of toxT. In this study, we show a near absenc...

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, Jan 24, 2015

The Journal of infectious diseases, 2015