Prevent Phage Adsorption Research Articles

Enterococcus faecalis Bacteriophage vB_EfaS_efap05-1 Targets the Surface Polysaccharide and ComEA Protein as the Receptors.

Enterococcus faecalis is a Gram-positive opportunistic pathogen that causes nosocomial infections in humans. Due to the growing threat of antibiotic resistance of E. faecalis, bacteriophage therapy is a promising option for treating of E. faecalis infection. Here, we characterized a lytic E. faecalis bacteriophage vB_EfaS_efap05-1 with a dsDNA genome of 56,563 bp. Phage vB_EfaS_efap05-1 had a prolate head and a tail, and belongs to Saphexavirus which is a member of Siphoviridae. Efap05-1 uses either surface polysaccharide or membrane protein ComEA as the receptor because the mutation of both genes (ComEA and UDP-glucose 4-epimerase galE) prevents phage adsorption and leads to phage resistance, and complementation of ComEA or galE could recover its phage sensitivity. Our results provided a comprehensive analysis of a new E. faecalis phage and suggest efap05-1 as a potential antimicrobial agent.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Gpwac of the T4-Type Bacteriophages: Structure, Function, and Evolution of a Segmented Coiled-Coil Protein That Controls Viral Infectivity

The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the approximately 50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.

Stable packaging of phage PRD1 DNA requires adsorption protein P2, which binds to the IncP plasmid-encoded conjugative transfer complex.

The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent K(d) of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor.

The Lactococcal Plasmid pNP40 Encodes a Third Bacteriophage Resistance Mechanism, One Which Affects Phage DNA Penetration

The lactococcal plasmid pNP40 mediates insensitivity to (phi)c2 by an early-acting phage resistance mechanism in addition to the previously identified abortive infection system, AbiF, in the Lactococcus lactis subsp. lactis MG1614 background. A second abortive infection determinant on pNP40, AbiE, does not confer resistance to (phi)c2. The early-acting mechanism on pNP40 does not prevent phage adsorption nor does it appear to operate by restriction/modification. Phage DNA was not detected in pNP40-containing cells until 30 min following exposure to (phi)c2 compared with 5 min in a sensitive host; however, electroporation of phage DNA into resistant hosts resulted in the release of phage progeny from a dramatically elevated number of cells compared with conventionally infected hosts. It appears therefore that pNP40 encodes a novel phage resistance mechanism which blocks DNA penetration specifically for (phi)c2.

Lactococcus lactis ssp. lactis C2 Bacteriophage sk1 Receptor involving Rhamnose and Glucose Moieties in the Cell Wall

Adsorption of bacteriophage sk1 to Lactococcus lactis ssp. lactis C2 was reduced 64, 26, 26, 5, and 8% by addition of rhamnose, glucose, galactose, N-acetylglucosamine, and fucose, respectively, to the adsorption assay. Lectins that were specific for N-acetylglucoseamine and galactose did not prevent phage adsorption. However, three lectins specific for glucose, but different in size, reduced phage adsorption from 0 to 50%. Another lectin, which was specific for rhamnose, reduced phage adsorption 80%. Rhamnose, but not glucose, partially inhibited phage infection when added to a growing culture. The data suggest that a glucose moiety is near the adsorption site or is an important conformational determinant of it.

Lytic bacteriophages ofStreptococcus mutans

Three phages ofStreptococcus mutans were obtained and partially characterized. The three phages, designated M102, e10, and f1, were found to be strictly lytic, with host ranges restricted to only serotype c, e, and f strains of this species, respectively. Phage sensitivity was not correlated with the presence of plasmids, at least in host strains of serotypes c and e. Each phage produced clear plaques in a number of standard media, even in the presence of sucrose, indicating that the extracellular glucan polysaccharides (mutan) produced by the hosts from this substrate do not prevent phage adsorption and growth. The phages were similar in size and morphology, having icosahedral heads and long (283-287 nm), flexible, noncontractile tails. The genome of each phage was found to consist of linear, double-stranded DNA, 31-35 kb in length, with a base composition of 37-38% G+C. Restricting phage DNAs with four enzymes produced fragment patterns unique to each phage, but common bands between M102 and e10 and between e10 and f1 were produced byBamHI. Labeled e10 and M102 DNAs hybridized strongly with all three phage DNAs, indicating that they share some common sequences. The three phages appear to be more similar than expected and probably evolved from a common ancestor.

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.

Characterization of Phage-Sensitive Mutants from a Phage-Insensitive Strain of Streptococcus lactis : Evidence for a Plasmid Determinant that Prevents Phage Adsorption

A phage-insensitive strain of Streptococcus lactis, designated ME2, was used as a prototype strain for the study of mechanisms and genetics of phage resistance in the lactic streptococci. Mutants sensitive to a Streptococcus cremoris phage, varphi18, were isolated at a level of 17% from cultures of ME2 after sequential transfer at 30 degrees C. Phage-sensitive mutants of ME2 were not fully permissive to varphi18. The efficiency of plating of varphi18 on the mutants was 5 x 10 as compared with <10 for varphi18 on ME2. Further characterization of the mutants showed that they efficiently adsorbed varphi18 at levels of >99.8%, whereas ME2 adsorbed only 20 to 40% of varphi18. These results suggest that increased phage susceptibility of the mutants may result from the loss of a mechanism that inhibits phage adsorption. Moreover, the high frequency of spontaneous mutation in ME2 indicates the involvement of an unstable genetic determinant in this phage defense mechanism. ME2 was shown to possess 13 plasmids ranging in size from 1.6 to 34 megadaltons. Of 40 mutants examined that had increased efficiencies of plating, all were missing a 30-megadalton plasmid, pME0030. These data suggest that pME0030 codes for a function that prevents phage adsorption. Further phenotypic characterization of the phage-sensitive mutants showed that some mutants were deficient in the ability to ferment lactose (Lac) and hydrolyze milk proteins (Prt). However, the Lac and Prt phenotype segregated independently of the phage-sensitivity phenotype. One phage-sensitive adsorption mutant, designated N1, was tested for susceptibility to 14 different phages. N1 showed increased capacity to adsorb 4 and to replicate 2 of these 14 phages, thereby indicating a phage resistance mechanism in ME2 that generalizes to phage interactions other than the specific varphi18-ME2 phage-host interaction. These data provide evidence for a unique plasmid-linked phage defense mechanism in phage-insensitive strains of lactic streptococci.

Function of T4D structural dihydrofolate reductase in bacteriophage infection.

Various properties of the bacteriophage structural dihydrofolate reductase (DFR) have been examined to determine its function during phage infection. It has been found that a binding site for reduced nicotinamide adenine dinucleotide phosphate (NADPH), most likely on the DFR present in the phage tail plate, is required for phage viability. Attachment of adenosine diphosphoribose, an analogue of NADPH, to this site prevents phage adsorption and injection. This adenosine diphosphoribose inhibition can be competitively reversed by the addition of NADPH or oxidized nicotinamide adenine dinucleotide phosphate. It is suggested that, during phage infection, the host bacterial cell might leak compounds functionally similar to the pyridine nucleotides. These compounds have been shown to nonenzymatically change the conformation of the phage tail plate DFR which is apparently necessary for successful injection.

Infection of competent Mycobacterium smegmatis with deoxyribonucleic acid extracted from bacteriophage B1.

A relatively competent state of Mycobacterium smegmatis for infection with deoxyribonucleic acid (DNA) extracted from phage B1 was found in the late log phase of bacterial growth. This state of the culture was used in quantitative studies on the infectivity of the DNA. The buoyant density of B1 DNA was 1.728 g/cc in CsCl, and 1 mug of the DNA produced 84 infective centers, the phage equivalent of which was 1.5 x 10(-8). The infectivity was destroyed by catalytic amounts of deoxyribonuclease but not by specific B1 antiserum. Tween 80, which prevents phage adsorption, did not prevent DNA infection. The response of plaque-forming ability to DNA concentration suggested that two or more molecules are required to initiate an infective center. The low efficiency of DNA infection in mycobacteria was considered to be caused by a limiting population of competent cells in the culture employed; in this experiment less than 10(-5) of the cells were infected with DNA. A typical cycle of infection was observed, although the latent period was prolonged and the burst size reduced after DNA infection. The transition of B1 DNA infection to deoxyribonuclease insensitivity had a lag period of about 10 min, and increased linearly with a velocity of about 0.24 infective centers per min per mug of DNA. Half of the infective titer was inactivated by heating at 92 C for 15 min. The melting temperature was about 96 C. Species barriers were not crossed by B1 DNA; however, the DNA was infectious for a B1-resistant mutant of the host.

INACTIVATION OF STREPTOCOCCAL BACTERIOPHAGE BY SULFHYDRYL REAGENTS.

SummaryAlthough there is evidence to suggest that cell wall components play a role as receptors for phage attachment to the cell, the details of virus adsorption and the prerequisite conditions for infection are not clearly elucidated. In the experiments reported here, it has been demonstrated that phages of Groups A and C streptococci are inactivated by certain sulfhydryl reagents such as PHMB and mercuric ion. The results suggest that these agents did not prevent phage adsorption but rather affected some later step in infection. The inactivation of phage infectivity by the sulfhydryl reagents was reversed by the thiol compound reduced glutathione, a finding consistent with the view that sulfhydryl groups of the phage tail may play a role in the early stages of phage infection.