Key Virulence Determinants Research Articles

Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine?

A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments. How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.

Application of Metagenomic Sequencing to Food Safety: Detection of Shiga Toxin-Producing Escherichia coli on Fresh Bagged Spinach

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection.

The complete genome sequence of Dickeya zeae EC1 reveals substantial divergence from other Dickeya strains and species.

BackgroundDickeya zeae is a bacterial species that infects monocotyledons and dicotyledons. Two antibiotic-like phytotoxins named zeamine and zeamine II were reported to play an important role in rice seed germination, and two genes associated with zeamines production, i.e., zmsA and zmsK, have been thoroughly characterized. However, other virulence factors and its molecular mechanisms of host specificity and pathogenesis are hardly known.ResultsThe complete genome of D. zeae strain EC1 isolated from diseased rice plants was sequenced, annotated, and compared with the genomes of other Dickeya spp.. The pathogen contains a chromosome of 4,532,364 bp with 4,154 predicted protein-coding genes. Comparative genomics analysis indicates that D. zeae EC1 is most co-linear with D. chrysanthemi Ech1591, most conserved with D. zeae Ech586 and least similar to D. paradisiaca Ech703. Substantial genomic rearrangement was revealed by comparing EC1 with Ech586 and Ech703. Most virulence genes were well-conserved in Dickeya strains except Ech703. Significantly, the zms gene cluster involved in biosynthesis of zeamines, which were shown previously as key virulence determinants, is present in D. zeae strains isolated from rice, and some D. solani strains, but absent in other Dickeya species and the D. zeae strains isolated from other plants or sources. In addition, a DNA fragment containing 9 genes associated with fatty acid biosynthesis was found inserted in the fli gene cluster encoding flagellar biosynthesis of strain EC1 and other two rice isolates but not in other strains. This gene cluster shares a high protein similarity to the fatty acid genes from Pantoea ananatis.ConlusionOur findings delineate the genetic background of D. zeae EC1, which infects both dicotyledons and monocotyledons, and suggest that D. zeae strains isolated from rice could be grouped into a distinct pathovar, i.e., D. zeae subsp. oryzae. In addition, the results of this study also unveiled that the zms gene cluster presented in the genomes of D. zeae rice isolates and D. solani strains, and the fatty acid genes inserted in the fli gene cluster of strain EC1 were likely derived from horizontal gene transfer during later stage of bacterial evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1545-x) contains supplementary material, which is available to authorized users.

Establishment of a mouse-adapted seasonal influenza A H3N2 virus strain and preliminary inquiry its molecular mechanism

Objective To establish adapted strain of seasonal influenza virus influenza A virus[A/Hong Kong/1968(H3N2)] in mice and explore the molecular mechanism of adaption preliminary. Methods The mice model was induced by nose dropping with wild type H3N2 influenza virus, by continuous passage in the lung of mice and detection of the clinical manifestation, viral load, virus titer, pathology, cytokines, to get the adapted strain of seasonal influenza A virus[A/Hong Kong/1968(H3N2)]. Results After 7 times continuous passage in the lung of mice, virulence was increased, viral load and virus titer were increased either. Genome sequencing and alignment indicated that the HA and NA gene was mutated. Conclusion The influenza virus H3N2 of mice lung adaption can be acquired from wild type seasonal H3N2 by continuous passage in the lung tissues of mice. Mutation of Ile to Val at residue 248 of HA and Val to Ile at residue 444of NA protein may be the key virulence determinant. Key words: Influenza A virus; Mice, inbread BALBC; Vircs cultivastion

Moderate PEGylation of the carrier protein improves the polysaccharide-specific immunogenicity of meningococcal group A polysaccharide conjugate vaccine

Neisseria meningitidis can cause severe and fulminant diseases such as meningitis. Meningococcal capsular polysaccharide (PS) is a key virulence determinant that is not able to induce immunological memory. Conjugation of PS to a carrier protein can significantly increase the immunogenicity of PS and induce immunological memory. Due to the classically described carrier-induced epitopic suppression (CIES) mechanisms, a strong immune response against the carrier protein could suppress the immune response to PS after coadministration of free carrier protein with the conjugate vaccine. However, it was not clear whether suppressing or enhancing the protein-specific immunogenicity could improve the PS-specific immunogenicity of the conjugate vaccine. Thus, moderate PEGylation, extensive PEGylation and oligomerization were used to regulate the immunogenicity of tetanus toxoid (TT) in the conjugate vaccine (PS-TT). Moderate PEGylation led to a 2.7-fold increase in the PS-specific IgG titers elicited by PS-TT. In contrast, extensive PEGylation and oligomerization of TT led to 1.4-fold and 1.6-fold decrease in the PS-specific IgG titers elicited by PS-TT, respectively. The PS-specific immunogenicity of PS-TT can be increased by moderate PEGylation through mild suppression of the TT-specific immunogenicity. The PS-specific immunogenicity of PS-TT was decreased through significant suppression or enhancement of the TT-specific immunogenicity. Thus, our study contributes to understand the CIES mechanisms and improve the PS-specific immunogenicity of a meningococcal PS conjugate vaccine.

Quantification of the APE2 gene expression level in Candida albicans clinical isolates from patients with diagnosed fungal infections.

The production of hydrolytic enzymes is considered a key virulence determinant of Candida albicans. Aminopeptidase 2 (Ape2) facilitates the penetration of C. albicans into the host tissue, by providing free amino acids to support fungal growth and proliferation. The objective of this study was to estimate the APE2 expression profile in C. albicans cells during invasion of the human epithelium. Sixty-one clinical fungal isolates and five reference strains were included in this study. The wild-type APE2 sequence was analyzed by polymerase chain reaction (PCR) amplification using genomic DNA from pathogenic isolates. Amplicons were verified in 1% agarose gel and visualized by illumination with ultraviolet (UV) light. The APE2 expression levels were analyzed with reverse transcription quantitative real-time PCR (RT-qPCR) and APE2 quantification was normalized against the reference gene in C. albicans cells grown in YEPD and during Caco-2 invasion. The APE2-specific PCR product band was found in all C. albicans and C. dubliniensis strains, but not in other common pathogenic fungi. APE2 transcript abundance was elevated in the clinical isolates growing on the Caco-2 cell line in comparison to their counterparts grown in YEPD. Our data indicate a potential role for Ape2 in the invasion of epithelial cells. APE2 expression is also strain-specific, and it is not related to isolation site or disease entity.

The endosomal sorting complex required for transport machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans.

Iron availability is a key determinant of virulence in the pathogenic fungus Cryptococcus neoformans. Previous work revealed that the ESCRT (endosomal sorting complex required for transport) protein Vps23 functions in iron acquisition, capsule formation and virulence. Here, we further characterized the ESCRT machinery to demonstrate that defects in the ESCRT-II and III complexes caused reduced capsule attachment, impaired growth on haem and resistance to non-iron metalloprotoporphyrins. The ESCRT mutants shared several phenotypes with a mutant lacking the pH-response regulator Rim101, and in other fungi, the ESCRT machinery is known to activate Rim101 via proteolytic cleavage. We therefore expressed a truncated and activated version of Rim101 in the ESCRT mutants and found that this allele restored capsule formation but not growth on haem, thus suggesting a Rim101-independent contribution to haem uptake. We also demonstrated that the ESCRT machinery acts downstream of the cAMP/protein kinase A pathway to influence capsule elaboration. Defects in the ESCRT components also attenuated virulence in macrophage survival assays and a mouse model of cryptococcosis to a greater extent than reported for loss of Rim101. Overall, these results indicate that the ESCRT complexes function in capsule elaboration, haem uptake and virulence via Rim101-dependent and independent mechanisms.

Deletion of a 77-base-pair inverted repeat element alters the synthesis of surface polysaccharides in Porphyromonas gingivalis.

Bacterial cell surface glycans, such as capsular polysaccharides and lipopolysaccharides (LPS), influence host recognition and are considered key virulence determinants. The periodontal pathogen Porphyromonas gingivalis is known to display at least three different types of surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown that PG0121 (in strain W83) encodes a DNABII histone-like protein and that this gene is transcriptionally linked to the K-antigen capsule synthesis genes, generating a large ∼19.4-kb transcript (PG0104-PG0121). Furthermore, production of capsule is deficient in a PG0121 mutant strain. In this study, we report on the identification of an antisense RNA (asRNA) molecule located within a 77-bp inverted repeat (77bpIR) element located near the 5' end of the locus. We show that overexpression of this asRNA decreases the amount of capsule produced, indicating that this asRNA can impact capsule synthesis in trans. We also demonstrate that deletion of the 77bpIR element and thereby synthesis of the large 19.4-kb transcript also diminishes, but does not eliminate, capsule synthesis. Surprisingly, LPS structures were also altered by deletion of the 77bpIR element, and reactivity to monoclonal antibodies specific to both O-LPS and A-LPS was eliminated. Additionally, reduced reactivity to these antibodies was also observed in a PG0106 mutant, indicating that this putative glycosyltransferase, which is required for capsule synthesis, is also involved in LPS synthesis in strain W83. We discuss our finding in the context of how DNABII proteins, an antisense RNA molecule, and the 77bpIR element may modulate expression of surface polysaccharides in P. gingivalis. The periodontal pathogen Porphyromonas gingivalis displays at least three different types of cell surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown using Northern analysis that the K-antigen capsule locus encodes a large transcript (∼19.4 kb), encompassing a 77-bp inverted repeat (77bpIR) element near the 5' end. Here, we report on the identification of an antisense RNA (asRNA) encoded within the 77bpIR. We show that overexpression of this asRNA or deletion of the element decreases the amount of capsule. LPS structures were also altered by deletion of the 77bpIR, and reactivity to monoclonal antibodies to both O-LPS and A-LPS was eliminated. Our data indicate that the 77bpIR element is involved in modulating both LPS and capsule synthesis in P. gingivalis.

You are what you secrete: extracellular proteins and virulence in Cryptococcus

Fungal organisms secrete a wide range of biomolecules, including degradative enzymes that are essential for nutrition, toxins, effectors and secondary compounds that modulate interactions with host animals and plants, and a variety of signaling and stress-related proteins. As these are likely to be key determinants of virulence and may also be useful diagnostic and therapeutic targets, we investigated the secretome of different strains of the fungal pathogen Cryptococcus. Virulent strains secreted predominantly hydrolytic and proteolytic enzymes, while the least virulent strain secreted a range of additional non-degradative proteins including many that lacked secretion signals, some that appear to be ‘moonlighting’, and a number that are known to be allergenic. It appears that in Cryptococcus, the secretome may influence virulence both through the presence of harmful enzymes and through the absence of proteins that alert the host defence mechanisms.

Back to the metal age: battle for metals at the host-pathogen interface during urinary tract infection.

Urinary tract infection (UTI) represents one of the most common bacterial infections in humans and uropathogenic E. coli (UPEC) is the major causative agent of UTI in people. Research on UPEC and other bacterial pathogens causing UTI has now identified the critical role of metal transport systems in the pathogenesis of UTI. Here we review the major effectors of metal transport in bacteria and host proteins that impair metal acquisition by bacterial pathogens. In particular, we describe the studies that identified iron, zinc and nickel import and copper export as key virulence and fitness determinants during UTI. Various metal transport systems and mechanisms that govern the expression of metal transport systems are also presented here. Specific examples from UPEC and other uropathogens, when available, are presented to depict the battle for metals at the host-pathogen interface during UTI.

A new regulator of pathogenicity (bvlR) is required for full virulence and tight microcolony formation in Pseudomonas aeruginosa.

LysR-type transcriptional regulators (LTTRs) are the most common family of transcriptional regulators found in the opportunistic pathogen Pseudomonas aeruginosa. They are known to regulate a wide variety of virulence determinants and have emerged recently as positive global regulators of pathogenicity in a broad spectrum of important bacterial pathogens. However, in spite of their key role in modulating expression of key virulence determinants underpinning pathogenic traits associated with the process of infection, surprisingly few are found to be transcriptionally altered by contact with host cells. BvlR (PA14_26880) an LTTR of previously unknown function, has been shown to be induced in response to host cell contact, and was therefore investigated for its potential role in virulence. BvlR expression was found to play a pivotal role in the regulation of acute virulence determinants such as type III secretion system and exotoxin A production. BvlR also played a key role in P. aeruginosa pathogenicity within the Caenorhabditis elegans acute model of infection. Loss of BvlR led to an inability to form tight microcolonies, a key step in biofilm formation in the cystic fibrosis lung, although surface attachment was increased. Unusually for LTTRs, BvlR was shown to exert its influence through the transcriptional repression of many genes, including the virulence-associated cupA and alg genes. This highlights the importance of BvlR as a new virulence regulator in P. aeruginosa with a central role in modulating key events in the pathogen-host interactome.

Screening of potent antibacterial agents targeting Clostridium difficile virulence factor toxin B: an in silico approach

Toxin B is the key determinant of virulence in Clostridium difficile. Cysteine protease domain (CPD) of toxin B plays a crucial role in host cell intoxication thereby making it a potential target for drug discovery. The present study is aimed at identifying the promising lead compounds targeting C. difficile toxin B CPD. Initial screening of the compounds was done using topomer search and drug-likeness properties. The subsequent molecular docking study yielded a set of lead compounds having better docking score and binding mode of interactions compared to the known inhibitor. Molecular dynamics simulations were performed to explore the stability of protein–ligand complexes. The identified promising lead molecules can be used for the development of therapeutics targeting C. difficile toxin B. Large scale screening protocol for identification of novel promising lead candidates against virulence factor (toxin B) of Clostridium difficile.

Secretory kinases in parasites block innate immunity (99.2)

Toxoplasma gondii is a widespread parasite of animals that is transmitted between cats and rodents, although it also infects a wide range of vertebrates including humans. Genetic mapping studies have identified a family of polymorphic serine threonine (S/T) kinases that control acute virulence in the mouse. These kinases are released from organelles called the rhoptries (ROP) and they target different host pathways. One of the key virulence determinants is ROP18, which phosphorylates switch region I in the GTPase domain of a family of immunity related GTPases, which are important in pathogen control. Phosphorylation in this key region prevents oligomerization and blocks GTPase function, thus protecting the parasite within the host cell. Recent studies have revealed that ROP18, and other active ROP kinases, are control by the pseudokinase ROP5, which lacks catalytic activity, but is required for acute virulence. ROP5 regulates the activity of ROP18, as well as binding to its substrates to facilitate their phosphorylation. Collectively, these studies reveal a complex network of S/T kinases in the parasite that control acute virulence of T. gondii by blocking innate immunity in the mouse.

A Biochemical Analysis of the Interaction of Porphyromonas gingivalis HU PG0121 Protein with DNA

K-antigen capsule, a key virulence determinant of the oral pathogen Porphyromonas gingivalis, is synthesized by proteins encoded in a series of genes transcribed as a large polycistronic message. Previously, we identified a 77-base pair inverted repeat region with the potential to form a large stem-loop structure at the 5′ end of this locus. PG0121, one of two genes flanking the capsule operon, was found to be co-transcribed with the operon and to share high similarity to the DNA binding protein HU from Escherichia coli. A null mutation in PG0121 results in down-regulation of transcription of the capsule synthesis genes and production of capsule. Furthermore, we have also shown that PG0121 gene can complement multiple deficiencies in a strain of E. coli that is deficient for both the alpha and beta subunits of HU. Here, we examined the biochemical properties of the interaction of PG0121 to DNA with the emphasis on the kinds of nucleic acid architectures that may be encountered at the 77-bp inverted repeat. We have concluded that although some DNA binding characteristics are shared with E. coli HU, HU PG0121 also shows some distinct characteristics that set it apart from other HU-like proteins tested to date. We discuss our results in the context of how PG0121 may affect the regulation of the K-antigen capsule expression.

Hyperexpression of α-hemolysin explains enhanced virulence of sequence type 93 community-associated methicillin-resistant Staphylococcus aureus

BackgroundThe community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined.ResultsHere, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus.ConclusionsThese data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

Extraction and Purification of Mycobacterial Mycolic Acids

Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that decorate the lipid. Mycolic acids play a critical role in the architecture and impermeability of the cell envelope, hence the natural resistance of mycobacteria to most antibiotic treatments. They are also key determinants of virulence in pathogenic species, including Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis. In addition, they are known as the primary target of several first-line and second-line antitubercular drugs. Thus, the unique enzymes involved in the mycolic acid biosynthetic pathway represent an attractive reservoir of targets for future chemotherapy whose developments are particularly warranted in the context of multi-drug-resistant and extensively-drug-resistant strains of M. tuberculosis. Herein, we describe a protocol to extract the mycolic acids from mycobacteria. Purification of the various subspecies may be particularly useful for subsequent structural studies involving mass spectrometry or NMR. The qualitative and quantitative biochemical characterization of the mycolic acid pattern by thin layer chromatography can be used to address how drugs alter mycolic acid biosynthesis (Alahari et al., 2007, Hartkoorn et al., 2012), to study the phenotypes of genetically modified mutants affected in this metabolic pathway (Bhatt et al., 2007) or to unravel new mycolic acid regulatory mechanisms (Vilcheze et al., 2014). The same protocol can be applied to all mycobacteria, including environmental and pathogenic species.

Phenol-soluble modulins

PSMs are a recently discovered family of short, amphipathic, α-helical peptides in staphylococci. Several PSMs are key virulence determinants, particularly in highly virulent Staphylococcus aureus strains. PSMα peptides of S. aureus facilitate neutrophil lysis after phagocytosis, and are key contributors to several infection types, including skin infection and bacteremia. Furthermore, all PSMs contribute to biofilm structuring and the dissemination of biofilm-associated infection. Cytolytic PSMs as produced by S. aureus appear to have evolved from original functions in the non-infectious lifestyle of staphylococci. The surfactant properties of PSMs, which they all share, are believed to facilitate growth on epithelial surfaces. The basic role of PSMs in staphylococcal physiology is underscored, for example, by their exceptionally strict and direct control by quorum-sensing and the presence of a dedicated secretion system. Targeting PSMs for anti-staphylococcal drug development may be a promising approach to overcome the problems associated with widespread antibiotic resistance in staphylococci.

S101 Pathogen associated molecular patterns in cystic fibrosis pathogens: Analysis of peptidoglycan structure

BackgroundPathogen associated molecular patterns (PAMPS) are key virulence determinants in triggering immune mediated damage in cystic fibrosis. The lipopolysaccharide (LPS) in Pseudomonas aeruginosa strains isolated from CF-lungs has “CF specific”...

Development of pneumococcal polysaccharide conjugate vaccine with long spacer arm

Streptococcus pneumoniae is a serious Gram-positive pathogen responsible for several life-threatening pneumococcal diseases. Pneumococcal capsular polysaccharide (CPS) is a key virulence determinant of S. pneumoniae and its immunogenicity can be improved by conjugation with a carrier protein. Reductive amination, the most widely used approach for pneumococcal CPS conjugate vaccine (PCV), suffers from low conjugation efficiency and the problem of steric hindrance. Here, copper-catalyzed azide-alkyne cycloaddition was used for development of PCV with long spacer arm (L-PCV). Tetanus toxoid (TT) was used as the carrier protein. The long spacer arm in L-PCV can minimize the problem of steric hindrance between CPS and TT, thereby improving the CPS-specific antibody titers in the mice model. L-PCV can also induce high avidity functional antibody and elicit immunological memory in response to the native CPS.

Isolation, characterization and comparative genomics of bacteriophage SfIV: a novel serotype converting phage from Shigella flexneri

BackgroundShigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful.ResultsIn this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host.ConclusionsThis study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri.