Gold Standard Cholera Diagnostics Are Tarnished by Lytic Bacteriophage and Antibiotics.

A fundamental, clinical, and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question, because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection and exposure to the first-line antibiotic azithromycin, detected in stool samples by mass spectrometry. Among diarrheal samples positive by nanoliter quantitative PCR (qPCR) for V. cholerae (n = 78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (odds ratio [OR], 0.108; 95% confidence interval [CI], 0.002 to 0.872) and 87% (OR, 0.130; 95% CI, 0.022 to 0.649), respectively, when lytic bacteriophage were detected. The odds that an RDT or qPCR was positive was reduced by more than 99% (OR, 0.00; 95% CI, 0.00 to 0.28) and 89% (OR, 0.11; 95% CI, 0.03 to 0.44), respectively, when azithromycin was detected. Analysis of additional samples from South Sudan found similar phage effects on RDTs; antibiotics were not assayed. Cholera burden estimates may improve by accommodating for the negative effects of lytic bacteriophage and antibiotic exposure on diagnostic positivity. One accommodation is using bacteriophage detection as a proxy for pathogen detection. These findings have relevance for other diagnostic settings where bacterial pathogens are vulnerable to lytic bacteriophage predation.

The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.

The numerous sigma (sigma) factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis sigma(B) regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.

The DosR regulon of M. tuberculosis and antibacterial tolerance.

Adaptation of Mycobacterium tuberculosis to an anaerobic dormant state that is tolerant to several antibacterials is mediated largely by a set of highly expressed genes controlled by DosR. A DosR mutant was constructed to investigate whether the DosR regulon is involved in antibacterial tolerance. We demonstrate that induction of the regulon is not required for drug tolerance either in vivo during a mouse infection or in vitro during anaerobic dormancy. Thus, drug tolerance observed in these models is due to other mechanisms such as the bacilli simply being in a non-replicating or low metabolic state. Our data also demonstrate that the DosR regulon is not essential for virulence during chronic murine infection. However, decreased lung pathology was observed in the DosR mutant. We also show that the DosR regulon genes are more highly conserved in environmental mycobacteria, than in pathogenic mycobacteria lacking a latent phase or environmental reservoir. It is possible that the DosR regulon could contribute to drug tolerance in human infections; however, it is not the only mechanism and not the primary mechanism for tolerance during a mouse infection. These data suggest that the regulon evolved not for pathogenesis or drug tolerance but for adaptation to anaerobic conditions in the environment and has been adapted by M. tuberculosis for survival during latent infection.

Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy.

The innate mechanisms used by Mycobacterium tuberculosis to persist during periods of non-proliferation are central to understanding the physiology of the bacilli during latent disease. We have used whole genome expression profiling to expose adaptive mechanisms initiated by M. tuberculosis in two common models of M. tuberculosis non-proliferation. The first of these models was a standard growth curve in which gene expression changes were followed from exponential growth through the transition to stationary phase. In the second model, we followed the adaptive process of M. tuberculosis during transition from aerobic growth to a state of anaerobic non-replicating persistence. The most striking finding from these experiments was the strong induction of the entire DosR "dormancy" regulon over approximately 20 days during the long transition to an anaerobic state. This is contrasted by the muted overall response to aerated stationary phase with only a partial dormancy regulon response. From the results presented here we conclude that the respiration-limited environment of the oxygen-depleted NRP model recreates at least one fundamental factor for which the genome of M. tuberculosis encodes a decisive adaptive program.

Regulation of the Mycobacterium tuberculosis PE/PPE genes.

The genome of Mycobacterium tuberculosis encodes approximately 170 members of the unique mycobacterial PE and PPE gene families. Evidence suggests members of these families are surface-associated cell wall proteins that may provide a diverse antigenic profile and affect immunity. To determine if the expression patterns of PE/PPE genes are consistent with a role in antigenic variability, we analyzed microarray data from 132 experimental conditions for expression of PE/PPE genes. Whole genome expression patterns show that the PE/PPE genes are regulated in a variable and largely independent manner. Gene expression profiling of 15 unique conditions identified differential regulation of 128 of the 169 PE/PPE genes. Expression of the PE/PPE genes appears to be controlled by a variety of independent mechanisms. These data indicate that differential expression of the PE/PPE genes has the potential to provide a dynamic antigenic profile during the course of changing microenvironments within the host.

The Mycobacterium tuberculosis ECF sigma factor sigmaE: role in global gene expression and survival in macrophages.

In previously published work, we identified three Mycobacterium tuberculosis sigma (sigma) factor genes responding to heat shock (sigB, sigE and sigH). Two of them (sigB and sigE) also responded to SDS exposure. As these responses to stress suggested that the sigma factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we started to define the sigmaE regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when sigmaE is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.

Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin.

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis alpha-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of vps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio cholerae O1 El Tor.

The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS(ETr)) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS(ETr) and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL, within the vps cluster of EPS(ETr) biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a positive regulator of vpsA and vpsL and thus acts to positively regulate EPS(ETr) production and biofilm formation.

Functional Genomics of Mycobacterium tuberculosis Using DNA Microarrays.

Completion of the sequence of the entire genome of strain H37Rv was a benchmark for Mycobacterium tuberculosis research (1). This achievement ushers in the era of genome-wide functional and comparative genomics for this organism. At present, the most powerful enabling technology of the postgenomic era is microarray-based hybridization. Microarrays, by whatever means they are fabricated, contain surface-bound representations of each open reading frame (ORF) of a sequenced genome. Thus, they provide a method for parallel sampling of thousands of different genes within a complex pool of nucleic acids. Microarray gene capacity readily accommodates the number of ORFs in the relatively small genomes of bacteria and yeast and, in principle, can accommodate the entire genetic repertoire of complex multicellular animals. Below, we discuss our fabrication and use of an M. tuberculosis microarray, containing representations of each of the identified 3924 ORFs of this organism. We will describe two applications of this method. In the first-microarray-based gene response, i.e., transcript profiling - we ask the question: which genes are selectively expressed under a particular condition of growth, in a particular host compartment or as a result of inhibition of a metabolic or biosynthetic pathway? In the second, comparative genomics, we use a microarray containing the ORFs of one strain or species to identify ORFs deleted or absent from a second strain or species whose genome sequence may not have been determined. In this manner, microarray-based comparative genomics seeks to learn the ORF-by-ORF relatedness of two similar, but nonidentical organisms whose biological differences are under investigation. Examples of each application have been applied to M. tuberculosis (2,3).

The complete genome sequence of Vibrio cholerae: a tale of two chromosomes and of two lifestyles.

Vibrio cholerae O1 has figured prominently in the history of infectious diseases as a cause of periodic global epidemics, an affliction of refugees in areas of social strife and as the disease first subjected to modern epidemiological analysis during the classic investigations of John Snow in mid-19th century London [1]. Thus, publication of the entire genome sequence of V. cholerae O1 (biotype El Tor) in Nature [2] by a consortium of investigators from The Institute for Genomic Research, the University of Maryland and Harvard Medical School is properly regarded as an historic event that will trigger a paradigm shift in the study of this organism.

Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization.

Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.

Complete DNA sequence and structural analysis of the enteropathogenic Escherichia coli adherence factor plasmid.

The complete nucleotide sequence and organization of the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid of EPEC strain B171 (O111:NM) were determined. The EAF plasmid encodes two known virulence-related operons, the bfp operon, which is composed of genes necessary for biosynthesis of bundle-forming pili, and the bfpTVW (perABC) operon, composed of regulatory genes required for bfp transcription and also for transcriptional activation of the eae gene in the LEE pathogenicity island on the EPEC chromosome. The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the bfp and bfpTVW (perABC) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements. Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E. coli. Another ORF, located between the bfp and bfpTVW operons, showed high similarity with trcA, a bfpT-regulated chaperone-like protein gene of EPEC. Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1). In addition, we identified a third region that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.

A novel chromosomal locus of enteropathogenic Escherichia coli (EPEC), which encodes a bfpT-regulated chaperone-like protein, TrcA, involved in microcolony formation by EPEC.

The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. After screening for promoters exhibiting bfpTVW-dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non-virulent E. coli laboratory strains, DH5alpha and HB101. Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E. coli K12. This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW-activated promoter was localized upstream of ORF1. An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription. An ORF1-His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone-like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene). The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment.

Comparative genomics of BCG vaccines by whole-genome DNA microarray.

Bacille Calmette-Guérin (BCG) vaccines are live attenuated strains of Mycobacterium bovis administered to prevent tuberculosis. To better understand the differences between M. tuberculosis, M. bovis, and the various BCG daughter strains, their genomic compositions were studied by performing comparative hybridization experiments on a DNA microarray. Regions deleted from BCG vaccines relative to the virulent M. tuberculosis H37Rv reference strain were confirmed by sequencing across the missing segment of the H37Rv genome. Eleven regions (encompassing 91 open reading frames) of H37Rv were found that were absent from one or more virulent strains of M. bovis. Five additional regions representing 38 open reading frames were present in M. bovis but absent from some or all BCG strains; this is evidence for the ongoing evolution of BCG strains since their original derivation. A precise understanding of the genetic differences between closely related Mycobacteria suggests rational approaches to the design of improved diagnostics and vaccines.

Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation.

The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease.

Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli.

Type IV bundle-forming pili of enteropathogenic Escherichia coli are required for the localized adherence and autoaggregation phenotypes. Whether these pili are also required for virulence was tested in volunteers by inactivating bfpA or bfpT (perA) encoding, respectively, the pilus subunit and the bfp operon transcriptional activator. Both mutants caused significantly less diarrhea. Mutation of the bfpF nucleotide-binding domain caused increased piliation, enhanced localized adherence, and abolished the twitching motility-dispersal phase of the autoaggregation phenotype. The bfpF mutant colonized the human intestine but was about 200-fold less virulent. Thus, BfpF is required for dispersal from the bacterial aggregate and for full virulence.

Construction and characterization of a partial Mycobacterium tuberculosis cDNA library of genes expressed at reduced oxygen tension.

To determine which bacterial genes could be expressed during tuberculosis in the human body, we have prepared and characterized a collection of cDNA clones corresponding to genes that are expressed by Mycobacterium tuberculosis during in vitro growth in 5% (v/v) oxygen. These cDNA clones were obtained by purifying total RNA from M. tuberculosis and cloning small cDNA segments into Escherichia coli followed by removal of clones containing ribosomal RNA sequences. From approx. 1700 clones, a collection of 170 clones containing non-ribosomal inserts were further characterized by PCR amplification. Inserts of more than 180bp were verified by Southern hybridization to have corresponding loci in M. tuberculosis genomic DNA and their sequence was determined. We describe the genes that have been identified using this approach. Multiple independent cDNA clones were obtained for two genes, one probably encoding a stable structural RNA and the other a homologue of ferritin. RNA levels for these two genes were monitored during growth at 20% oxygen, 5% oxygen and in the nearly anaerobic culture sediments. No difference in expression levels was found at 5% oxygen compared to 20% oxygen. RNA levels for the ferritin homologue gene were significantly lower in culture sediments. The stable structural RNA, however, showed very high expression levels independently of culture conditions.