Phage-encoded Virulence Factor Research Articles

Targeted proteomics links virulence factor expression with clinical severity in staphylococcal pneumonia.

The bacterial pathogen Staphylococcus aureus harbors numerous virulence factors that impact infection severity. Beyond virulence gene presence or absence, the expression level of virulence proteins is known to vary across S. aureus lineages and isolates. However, the impact of expression level on severity is poorly understood due to the lack of high-throughput quantification methods of virulence proteins. We present a targeted proteomic approach able to monitor 42 staphylococcal proteins in a single experiment. Using this approach, we compared the quantitative virulomes of 136 S. aureus isolates from a nationwide cohort of French patients with severe community-acquired staphylococcal pneumonia, all requiring intensive care. We used multivariable regression models adjusted for patient baseline health (Charlson comorbidity score) to identify the virulence factors whose in vitro expression level predicted pneumonia severity markers, namely leukopenia and hemoptysis, as well as patient survival. We found that leukopenia was predicted by higher expression of HlgB, Nuc, and Tsst-1 and lower expression of BlaI and HlgC, while hemoptysis was predicted by higher expression of BlaZ and HlgB and lower expression of HlgC. Strikingly, mortality was independently predicted in a dose-dependent fashion by a single phage-encoded virulence factor, the Panton-Valentine leucocidin (PVL), both in logistic (OR 1.28; 95%CI[1.02;1.60]) and survival (HR 1.15; 95%CI[1.02;1.30]) regression models. These findings demonstrate that the in vitro expression level of virulence factors can be correlated with infection severity using targeted proteomics, a method that may be adapted to other bacterial pathogens.

Dissemination of a phage-encoded virulence factor in a pandemic S. Typhimurium

Salmonella Typhimurium is the second most common cause of foodborne salmonellosis. Phage-mediated horizontal gene transfer contributes to the virulence of S. Typhimurium. An example of a phage-encoded virulence gene is sopE, a T3SS effector, found rarely in Typhimurium and associated with epidemics. The current pandemic and multi-drug resistant monophasic variant of S. Typhimurium (S. 4,[5],12:i:-) acquired sopE in multiple events following the lysogeny of a previously undescribed bacteriophage, mTmV. The current study aimed to further investigate the association of the virulence gene with S. 4,[5],12:i:- and assess its epidemiological impact. To this end, a large collection of clinical S. Typhimurium isolates from the UK have been analysed for the sopE gene and mTmV presence using a phylogenomic approach. While a large proportion of S. 4,[5],12:i:- (41 %) carried the sopE gene, few isolates outside the epidemic clade harboured it. Notably, the mTmV bacteriophage was identified only in S. 4,[5],12:i:-, although laboratory experiments demonstrated that the phage host range is not restricted to it. Nonetheless, we identified the phage in other S. enterica serovars circulating in the same ecological niche of S. 4,[5],12:i:-. In addition, a genomic characterisation of mTmV was performed revealing an unexpected level of phage variation. Finally, we identified a novel phage-like element harbouring the gene. The study revealed the large dissemination and selection of the virulence gene in the current epidemic, which is mobilised by multiple and distinct mobile genetic elements.

Identification of PblB mediating galactose-specific adhesion in a successful Streptococcus pneumoniae clone.

The pneumococcal genome is variable and there are minimal data on the influence of the accessory genome on phenotype. Pneumococcal serotype 14 sequence type (ST) 46 had been the most prevalent clone causing pneumonia in children in Taiwan. A microarray was constructed using the genomic DNA of a clinical strain (NTUH-P15) of serotype 14 ST46. Using DNA hybridization, genomic variations in NTUH-P15 were compared to those of 3 control strains. Microarray analysis identified 7 genomic regions that had significant increases in hybridization signals in the NTUH-P15 strain compared to control strains. One of these regions encoded PblB, a phage-encoded virulence factor implicated (in Streptococcus mitis) in infective endocarditis. The isogenic pblB mutant decreased adherence to A549 human lung epithelial cell compared to wild-type NTUH-P15 strain (P = 0.01). Complementation with pblB restored the adherence. PblB is predicted to contain a galactose-binding domain-like region. Preincubation of NTUH-P15 with D-galactose resulted in decreases of adherence to A549 cell in a dose-dependent manner. Challenge of mice with NTUH-P15, isogenic pblB mutant and pblB complementation strains determined that PblB was required for bacterial persistence in the nasopharynx and lung. PblB, as an adhesin mediating the galactose-specific adhesion activity of pneumococci, promote pneumococcal clonal success.

Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for beta-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a phi13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of phi13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim.

Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

Why Bacteriophage Encode Exotoxins and other Virulence Factors

This study considers gene location within bacteria as a function of genetic element mobility. Our emphasis is on prophage encoding of bacterial virulence factors (VFs). At least four mechanisms potentially contribute to phage encoding of bacterial VFs: (i) Enhanced gene mobility could result in greater VF gene representation within bacterial populations. We question, though, why certain genes but not others might benefit from this mobility. (ii) Epistatic interactions—between VF genes and phage genes that enhance VF utility to bacteria—could maintain phage genes via selection acting on individual, VF-expressing bacteria. However, is this mechanism sufficient to maintain the rest of phage genomes or, without gene co-regulation, even genetic linkage between phage and VF genes? (iii) Phage could amplify VFs during disease progression by carrying them to otherwise commensal bacteria colocated within the same environment. However, lytic phage kill bacteria, thus requiring assumptions of inclusive fitness within bacterial populations to explain retention of phage-mediated VF amplification for the sake of bacterial utility. Finally, (iv) phage-encoded VFs could enhance phage Darwinian fitness, particularly by acting as ecosystem-modifying agents. That is, VF-supplied nutrients could enhance phage growth by increasing the density or by improving the physiology of phage-susceptible bacteria. Alternatively, VF-mediated break down of diffusion-inhibiting spatial structure found within the multicellular bodies of host organisms could augment phage dissemination to new bacteria or to environments. Such phage-fitness enhancing mechanisms could apply particularly given VF expression within microbiologically heterogeneous environments, ie, ones where phage have some reasonable potential to acquire phage-susceptible bacteria.

Transcription of the Toxin Genes Present within the Staphylococcal Phage φSa3ms Is Intimately Linked with the Phage's Life Cycle

phiSa3ms, a lysogenic bacteriophage encoding the staphylococcal enterotoxins SEA, SEG, and SEK and the fibrinolytic enzyme staphylokinase (Sak), was identified in the unannotated genome sequence of the hypervirulent community-acquired Staphylococcus aureus strain 476. We found that mitomycin C induction of phiSa3ms led to increased transcription of all four virulence factors. The increase in sea and sak transcription was a result of read-through transcription from upstream latent phage promoters and an increase in phage copy number. The majority of the seg2 and sek2 transcripts were shown to initiate from the upstream phage cI promoter and hence were regulated by factors influencing cI transcription. The lysogeny module of phiSa3ms was shown to have some lambda-like features with divergent cI and cro genes. Band shift assays were used to identify binding sites for both CI and Cro within the region between these genes, suggesting a mechanism of control for the phiSa3ms lytic-lysogenic switch. Our findings suggest that the production of phage-encoded virulence factors in S. aureus may be regulated by processes that govern lysogeny.

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.

Sequence analysis of the Mycoplasma arthritidis bacteriophage MAV1 genome identifies the putative virulence factor.

The bacteriophage MAV1 is required for the development of arthritis in rats after infection with its host Mycoplasma arthritidis. To identify the phage-encoded virulence factor for this arthritis, the complete nucleotide sequence of MAV1 was determined. The linear double-stranded genome of MAV1 is 15644bp and contains 15 ORFs. Putative protein products from these ORFs were identified by comparison of the deduced amino acid sequences to known proteins and comprise DNA replication, restriction-modification, structural, regulatory, and integration/excision proteins. Eight putative promoters were identified; four of these would produce polycistronic transcripts. Translation of each ORF appears to be initiated independently, with each having its own RBS. A single ORF, vir, was identified on the minus strand of the phage genome. The putative protein product of vir contains a classic prokaryotic lipoprotein signal sequence and is a strong candidate for the MAV1-encoded virulence determinant.