Matthew K. Waldor

Lysogenic Conversion by a Filamentous Phage Encoding Cholera Toxin

Vibrio cholerae, the causative agent of cholera, requires two coordinately regulated factors for full virulence: cholera toxin (CT), a potent enterotoxin, and toxin-coregulated pili (TCP), surface organelles required for intestinal colonization. The structural genes for CT are shown here to be encoded by a filamentous bacteriophage (designated CTXΦ), which is related to coliphage M13. The CTXΦ genome chromosomally integrated or replicated as a plasmid. CTXΦ used TCP as its receptor and infected V. cholerae cells within the gastrointestinal tracts of mice more efficiently than under laboratory conditions. Thus, the emergence of toxigenic V. cholerae involves horizontal gene transfer that may depend on in vivo gene expression.

Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention

Experimental allergic encephalomyelitis (EAE) is an induced autoimmune disease mediated by CD4+ T lymphocytes. Analysis of T cell receptors of myelin basic protein-specific encephalitogenic T cell clones derived from six different PL/J (H-2u) or (PL/J x SJL) F1 (H-2uxs) mice revealed a limited heterogeneity in primary structure. In vivo, the majority of T lymphocytes recognize the N-terminal MBP-nonapeptide in association with I-Au and utilize the V beta 8 gene element. cDNA-sequencing showed that all T cell receptors from a panel of such T cell clones, grown in vitro, share the same V alpha gene segment. Despite heterogeneity in the D-J regions, the clones unexpectedly display a striking similarity in fine specificity. Based on these results, prevention and reversal of autoimmune disease with V beta 8-specific monoclonal antibodies was achieved.

Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany.

BACKGROUND A large outbreak of diarrhea and the hemolytic-uremic syndrome caused by an unusual serotype of Shiga-toxin-producing Escherichia coli (O104:H4) began in Germany in May 2011. As of July 22, a large number of cases of diarrhea caused by Shiga-toxin-producing E. coli have been reported--3167 without the hemolytic-uremic syndrome (16 deaths) and 908 with the hemolytic-uremic syndrome (34 deaths)--indicating that this strain is notably more virulent than most of the Shiga-toxin-producing E. coli strains. Preliminary genetic characterization of the outbreak strain suggested that, unlike most of these strains, it should be classified within the enteroaggregative pathotype of E. coli. METHODS We used third-generation, single-molecule, real-time DNA sequencing to determine the complete genome sequence of the German outbreak strain, as well as the genome sequences of seven diarrhea-associated enteroaggregative E. coli serotype O104:H4 strains from Africa and four enteroaggregative E. coli reference strains belonging to other serotypes. Genomewide comparisons were performed with the use of these enteroaggregative E. coli genomes, as well as those of 40 previously sequenced E. coli isolates. RESULTS The enteroaggregative E. coli O104:H4 strains are closely related and form a distinct clade among E. coli and enteroaggregative E. coli strains. However, the genome of the German outbreak strain can be distinguished from those of other O104:H4 strains because it contains a prophage encoding Shiga toxin 2 and a distinct set of additional virulence and antibiotic-resistance factors. CONCLUSIONS Our findings suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak. More broadly, these findings highlight the way in which the plasticity of bacterial genomes facilitates the emergence of new pathogens.

SOS response promotes horizontal dissemination of antibiotic resistance genes

Mobile genetic elements have a crucial role in spreading antibiotic resistance genes among bacterial populations. Environmental and genetic factors that regulate conjugative transfer of antibiotic resistance genes in bacterial populations are largely unknown. Integrating conjugative elements (ICEs) are a diverse group of mobile elements that are transferred by means of cell–cell contact and integrate into the chromosome of the new host. SXT is a ∼100-kilobase ICE derived from Vibrio cholerae that encodes genes that confer resistance to chloramphenicol, sulphamethoxazole, trimethoprim and streptomycin. SXT-related elements were not detected in V. cholerae before 1993 but are now present in almost all clinical V. cholerae isolates from Asia. ICEs related to SXT are also present in several other bacterial species and encode a variety of antibiotic and heavy metal resistance genes. Here we show that SetR, an SXT encoded repressor, represses the expression of activators of SXT transfer. The ‘SOS response’ to DNA damage alleviates this repression, increasing the expression of genes necessary for SXT transfer and hence the frequency of transfer. SOS is induced by a variety of environmental factors and antibiotics, for example ciprofloxacin, and we show that ciprofloxacin induces SXT transfer as well. Thus, we present a mechanism by which therapeutic agents can promote the spread of antibiotic resistance genes.

The Origin of the Haitian Cholera Outbreak Strain

BACKGROUND Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti. METHODS We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of 23 diverse strains of V. cholerae to assess the likely origin of the cholera outbreak in Haiti. RESULTS Both single-nucleotide variations and the presence and structure of hypervariable chromosomal elements indicate that there is a close relationship between the Haitian isolates and variant V. cholerae El Tor O1 strains isolated in Bangladesh in 2002 and 2008. In contrast, analysis of genomic variation of the Haitian isolates reveals a more distant relationship with circulating South American isolates. CONCLUSIONS The Haitian epidemic is probably the result of the introduction, through human activity, of a V. cholerae strain from a distant geographic source. (Funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.).

Quinolone Antibiotics Induce Shiga Toxin-Encoding Bacteriophages, Toxin Production, and Death in Mice

Shiga toxin-producing Escherichia coli (STEC) cause significant disease; treatment is supportive and antibiotic use is controversial. Ciprofloxacin but not fosfomycin causes Shiga toxin-encoding bacteriophage induction and enhanced Shiga toxin (Stx) production from E. coli O157:H7 in vitro. The potential clinical relevance of this was examined in mice colonized with E. coli O157:H7 and given either ciprofloxacin or fosfomycin. Both antibiotics caused a reduction in fecal STEC. However, animals treated with ciprofloxacin had a marked increase in free fecal Stx, associated with death in two-thirds of the mice, whereas fosfomycin did not. Experiments that used a kanamycin-marked Stx2 prophage demonstrated that ciprofloxacin, but not fosfomycin, caused enhanced intraintestinal transfer of Stx2 prophage from one E. coli to another. These observations suggest that treatment of human STEC infection with bacteriophage-inducing antibiotics, such as fluoroquinolones, may have significant adverse clinical consequences and that fluoroquinolone antibiotics may enhance the movement of virulence factors in vivo.

Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow

Integrative and conjugative elements (ICEs) are a diverse group of mobile genetic elements found in both Gram-positive and Gram-negative bacteria. These elements primarily reside in a host chromosome but retain the ability to excise and to transfer by conjugation. Although ICEs use a range of mechanisms to promote their core functions of integration, excision, transfer and regulation, there are common features that unify the group. This Review compares and contrasts the core functions for some of the well-studied ICEs and discusses them in the broader context of mobile-element and genome evolution.

Bacteriophage control of bacterial virulence.

In 1930, Felix d’Herelle wrote “. . .the actions and reactions are not solely between these two beings, man and bacterium, for the bacteriophage also intervenes; a third living being and, hence, a third variable is introduced” (19). The contribution of bacteriophages to the pathogenicity of their bacterial hosts began to be uncovered as early as 1927, when Frobisher and Brown discovered that nontoxigenic streptococci exposed to filtered supernatants of toxigenic streptococcal cultures acquired the ability to produce scarlatinal toxin (28). We now know that these supernatants contained a bacteriophage encoding the scarlatinal toxin and that these investigators were describing transduction, i.e., the transfer of genetic material to a bacterial cell via phage infection. Although these early investigators lacked a mechanistic explanation for their observations, they postulated that the bacterium was “within certain limits, perhaps a matter of secondary importance and that toxicogenicity might be a property that could be acquired by different types of organisms.” (28). Their hypothesis that bacteria acquire virulence properties has since gained widespread acceptance, as many virulence genes have been shown to undergo transfer among bacteria by phages (via transduction) and other mobile genetic elements such as plasmids (via conjugation). Over time, a number of toxin genes were found to be phage encoded, and consideration of the role of phages in bacterial pathogenesis emphasized the dissemination of toxin genes among bacterial strains (10). However, it has become increasingly clear that toxin genes are only a subset of the diverse virulence factors encoded by bacteriophages. For example, some phages encode regulatory factors that increase expression of virulence genes not encoded by the phage (84), while others encode enzymes that alter bacterial components related to virulence (31, 58). Furthermore, phages have unique properties that enable them to contribute more directly to bacterial virulence than via transduction. Structural components of virion particles, for example, may be directly pathogenic (5, 6, 99). Additionally, phage-encoded genes frequently undergo replication and transcriptional activation following prophage induction, a process that was speculated to have a role in the production of diphtheria toxin by Corynebacterium diphtheriae as early as 1960 (3). Since d’Herelle’s time, his notion of the phage as a third variable in bacterial pathogenesis has proven correct. However, while d’Herelle emphasized the diminution of bacterial virulence by bacteriophages (19), we have instead come to learn that phages serve as a driving force in bacterial pathogenesis, acting not only in the evolution of bacterial pathogens through gene transfer, but also contributing directly to bacterial pathogenesis at the time of infection. This review provides a discussion of (i) the discovery of phage-encoded virulence factors, (ii) bacterial virulence properties altered by phages, (iii) regulation of phage-encoded virulence factors, and (iv) the role of in situ prophage induction in the control of bacterial virulence.

Targeting QseC Signaling and Virulence for Antibiotic Development

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.

D-amino acids govern stationary phase cell wall remodeling in bacteria

Anyone for d? The chemistry of amino acids comes in two chirally distinct flavors—so-called l- and d-enantiomers. By far the most commonly used form of amino acids in all kingdoms of life is the l-form. Now Lam et al. (p. 1552; see the Perspective by Blanke) present the unanticipated observation that diverse bacteria release large amounts of various d-amino acids into the environment in a population density–dependent fashion and that d-amino acids act as extracellular effectors that regulate the composition, structure, amount, and strength of peptidoglycan, the major stress-bearing component of the bacterial cell wall. Bacteria produce D-amino acids to regulate their cell wall composition, structure, amount, and strength. In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.