AbiA,a lactococcal abortive infection mechanism functioning in Streptococcus thermophilus

The lactococcal abortive infection mechanisms AbiA and AbiG were introduced into Streptococcus thermophilus 4035, and a range of phages capable of infecting this host were examined for sensitivity to these mechanisms. AbiA proved effective against six phages when examined at a growth temperature of 30 degrees C but had no effect on any of the phages when tested at 37 or 42 degrees C. AbiG failed to affect any of the S. thermophilus phages at 30, 37, or 42 degrees C.     

Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40.

The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.     

Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis

During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10(-5) and 10(-7)) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.     

Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infection

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis.     

The vir gene of bacteriophage MAV1 confers resistance to phage infection on Mycoplasma arthritidis

Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn4001T and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all vir-containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.     

Lactococcus lactis AbiD1 abortive infection efficiency is drastically increased by a phage protein

Three genetically defined Actinobacillus pleuropneumoniae serotype 7 mutants with deletions in the small (tbpB), the large (tbpA), and both transferrin binding protein genes were constructed and examined in an aerosol infection model. Neither mutant caused clinical disease or could be reisolated, and no immune response could be detected 21 days after infection. This result clearly implies that each transferrin binding protein on its own is a virulence factor of A. pleuropneumoniae serotype 7.     

Identification of a recA homolog (recALP) on the conjugative lactococcal phage resistance plasmid pNP40: evidence of a role for chromosomally encoded recAL in abortive infection.

The determinants for two bacteriophage resistance mechanisms, AbiE and AbiF, are separated by approximately 3,300 nucleotides on the lactococcal plasmid pNP40 (P. Garvey, G.F. Fitzgerald, and C. Hill, Appl. Environ. Microbiol. 61:4321-4328, 1995). DNA sequence analysis of the intervening region led to the identification of two open reading frames (ORFs) which are transcribed in the opposite direction to the Abi determinants. One of these ORFs encodes a recA homolog (designated recALP). This is the first report of a recA-like determinant located to a plasmid. The second ORF (orfU) shares homology with the umuC gene of the SOS response. Analysis of a number of lactococcal strains confirmed the presence of recALP-like sequences in at least two other lactococcal strains. The proximity of the recA and umuC homologs suggested a possible role in the phase resistance encoded by the Abi determinants. However, no evidence was obtained to demonstrate a function for either ORF in the expression of either AbiE or AbiF. Nor could the recALP gene restore resistance to mitomycin in a recA-deficient lactococcal strain, VEL1122. Interestingly, it was shown that the chromosomally encoded recA is necessary for complete expression of the AbiF phenotype, confirming a role for RecA in this abortive infection system.     

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Abstract Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac⁺) to a plasmid-free Lac⁻ L. lactis strain. In each case, some Lac⁺ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra⁺), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.     

A topological model of the baseplate of lactococcal phage Tuc2009

Phages infecting Lactococcus lactis, a Gram-positive bacterium, are a recurrent problem in the dairy industry. Despite their economical importance, the knowledge on these phages, belonging mostly to Siphoviridae, lags behind that accumulated for members of Myoviridae. The three-dimensional structures of the receptor-binding proteins (RBP) of three lactococcal phages have been determined recently, illustrating their modular assembly and assigning the nature of their bacterial receptor. These RBPs are attached to the baseplate, a large phage organelle, located at the tip of the tail. Tuc2009 baseplate is formed by the products of 6 open read frames, including the RBP. Because phage binding to its receptor induces DNA release, it has been postulated that the baseplate might be the trigger for DNA injection. We embarked on a structural study of the lactococcal phages baseplate, ultimately to gain insight into the triggering mechanism following receptor binding. Structural features of the Tuc2009 baseplate were established using size exclusion chromatography coupled to on-line UV-visible absorbance, light scattering, and refractive index detection (MALS/UV/RI). Combining the results of this approach with literature data led us to propose a "low resolution" model of Tuc2009 baseplate. This model will serve as a knowledge base to submit relevant complexes to crystallization trials.     

Identification of adsorption inhibition, restriction/modification and abortive infection type phage resistance systems in Lactococcus lactis strains

98 Lactococcus lactis strains were isolated from traditional fermented milk products in Turkey tested against 60 lactococcal lytic phages to determine their resistance levels. While 82 L. lactis strains were sensitive against lactic phages at different levels, 16 L. lactis strains showed resistance to all phages tested. Types of phage resistance among 16 L. lactis strains were identified as phage adsorption inhibition in eight strains, restriction/modification in six strains and abortive infection (heat sensitive phage resistance) in two strains, using three broad-spectrum phages phi pll 98-32, phi pld 67-42 and phi pld 67-44.     

A nucleic acid hydrolyzing recombinant antibody confers resistance to curtovirus infection in tobacco

A catalytic single chain variable antibody (scFv), 3D8 scFv, which has DNase activities, was functionally expressed in Nicotiana tabacum. The subcellular localization of the GFP-fused 3D8 indicated that the 3D8 protein was expressed in the cytosol of the N. tabacum protoplasts. Progenies of the transgenic tobacco plants exhibited complete resistance against two single stranded (ss) DNA geminiviruses, including the Beet curly top virus and the Beet severe curly top virus, without viral accumulation or disease symptoms. We presented a novel strategy for targeting the viral DNA itself in a sequence non-specific manner, rather than the viral proteins or RNAs, in order to generate virus-resistant transgenic plants. No noticeable adverse effects on the growth and reproduction of the transgenic plants were observed. Our results demonstrated that targeting viral DNA is an effective strategy for protecting plants from ssDNA viruses.     

A common polymorphism in TLR3 confers natural resistance to HIV-1 infection

TLR3 recognizes dsRNA and activates antiviral immune responses through the production of inflammatory cytokines and type I IFNs. Genetic association studies have provided evidence concerning the role of a polymorphism in TLR3 (rs3775291, Leu412Phe) in viral infection susceptibility. We genotyped rs3775291 in a population of Spanish HIV-1-exposed seronegative (HESN) individuals who remain HIV seronegative despite repeated exposure through i.v. injection drug use (IDU-HESN individuals) as witnessed by their hepatitis C virus seropositivity. The frequency of individuals carrying at least one 412Phe allele was significantly higher in IDU-HESN individuals compared with that of a matched control sample (odds ratio for a dominant model = 1.87; 95% confidence interval, 1.06-3.34; p = 0.023). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Similar results were obtained: the frequency of individuals carrying at least one 412Phe allele was significantly higher compared with that of a matched control sample (odds ratio, 1.79; 95% confidence interval, 1.05-3.08; p = 0.029). In vitro infection assays showed that in PBMCs carrying the 412Phe allele, HIV-1(Ba-L) replication was significantly reduced (p = 0.025) compared with that of Leu/Leu homozygous samples and was associated with a higher expression of factors suggestive of a state of immune activation (IL-6, CCL3, CD69). Similarly, stimulation of PBMCs with a TLR3 agonist indicated that the presence of the 412Phe allele results in a significantly increased expression of CD69 and higher production of proinflammatory cytokines including IL-6 and CCL3. The data of this study indicate that a common TLR3 allele confers immunologically mediated protection from HIV-1 and suggest the potential use of TLR3 triggering in HIV-1 immunotherapy.     

Reprogramming the infection mechanism of a filamentous phage

When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.