SummaryPrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA regulon, we constructed a whole-genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild-type EGDe and a prfA -deleted mutant (EGDe D D D D prfA ). Both strains were grown at 37 ∞ ∞ ∞ ∞ C in brain-heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788 , both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes ( lmo0178-lmo0184 ) are organized in an operon. Group III comprises 53 genes, of which only two ( lmo0596 and lmo2067 ) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14 prfA *) and its corresponding prfA -deleted mutant (P14 D D D D prfA ) and the EGDe strain revealed interesting strain-specific differences. Sequences strongly similar to a sigma Bdependent promoter were identified upstream of 22 group III genes. These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A-dependent promoter. In contrast, a second set of PrfA-regulated genes lack a PrfA box and are expressed from a sigma B-dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.

SummaryPrfA, a transcription factor structurally related to Crp/ Fnr, activates Listeria monocytogenes virulence genes during intracellular infection. We report two new PrfA* mutations causing the constitutive overexpression of the PrfA regulon. Leu-140Phe lies in a a a a D adjacent to the DNA-binding motif in the C-terminal domain, like a previously characterized PrfA* mutation (Gly-145Ser). Ile-45Ser, in contrast, maps to the N-terminal b b b b -roll, a structure similar to that of the Crp cAMP binding site. The in vitro transcriptional properties of recombinant PrfA* I45S and PrfA* G145S were compared to those of PrfA WT at two differentially regulated PrfA-dependent promoters, P plcA and P actA . The two PrfA* mutations increased the affinity for the target DNA to a different extent, and the differences in DNA binding (PrfA* G145S > PrfA* I45S >>> PrfA WT ) correlated with proportional differences in transcriptional activity. The use of the PrfA* proteins revealed that P plcA had a greater affinity for, and was more sensitive to, PrfA than P actA . RNA polymerase (RNAP) initiated transcription independently of PrfA at P plcA , but not at P actA , consistent with bandshift experiments suggesting that P plcA has a greater affinity for RNAP than P actA . Thus, differences in affinity for both PrfA and RNAP appear to determine the different expression pattern of PrfA-regulated promoters. Modelling of the PrfA* mutations in the crystal structure of PrfA and comparison with structure-function analyses of Crp, in which similar mutations lead to constitutively active (cAMPindependent) Crp* proteins, suggested that PrfA shares with Crp an analogous mechanism of cofactormediated allosteric shift. Our data support a regulatory model in which changes in PrfA-dependent gene expression are primarily accounted for by changes in PrfA activity.

The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel‐shaped co‐operative haemolytic (‘CAMP‐like’) reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (β‐toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low‐stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock‐out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel‐shaped CAMP‐like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial‐like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane‐damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane‐damaging activity that, together with PlcA and PlcB, may act in concert with the pore‐forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5′ end of smcL is contiguous with the internalin locus i‐inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.

We assessed the role of the actin‐polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes. An in frame ΔactA mutant was constructed in a hyperinvasive strain of prfA* genotype, in which all genes of the PrfA‐dependent virulence regulon, including actA, are highly expressed in vitro. Loss of ActA production in prfA* bacteria reduced entry into Caco‐2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the ΔactA prfA* mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS‐1 fibroblasts and Hepa 1‐6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non‐invasive species into epithelial cell lines, but not into COS‐1 and Hepa 1‐6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco‐2 human enterocytes suggested that this pathway involves microvilli. prfA* bacteria, but not wild‐type bacteria (which express PrfA‐dependent genes very weakly in vitro) or prfA*ΔactA bacteria, efficiently invaded differentiated Caco‐2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA* strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin‐based motility and host cell tropism and invasion.

SummaryVirulence genes from the facultative intracellular pathogen Listeria mono cytogenes are controlled by the transcriptional regulator PrfA. Although PrfA synthesis is activated at 37 ∞ ∞ ∞ ∞ C, PrfA-dependent expression remains low in rich medium. However, a strong induction of the PrfA regulon is observed when L. monocytogenes is cultured in the presence of activated charcoal. Here, we show that the 'charcoal effect' results from the adsorption of a diffusible autorepressor substance released by L. monocytogenes during exponential growth. Analyses using an L. monocytogenes strain in which the prfA gene is expressed constitutively at 37 ∞ ∞ ∞ ∞ C from a plasmid indicate that the autoregulatory substance represses PrfA-dependent expression by inhibiting PrfA activity. PrfA presumably functions via an allosteric activation mechanism. The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA. Our data indicate that the listerial autorepressor is a low-molecular-weight hydrophobic substance. We suggest that this diffusible substance mediates a quorum-sensing mechanism by which L. monocytogenes restricts the expression of its PrfA virulence regulon. This autoregulatory pathway could serve L. monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37 ∞ ∞ ∞ ∞ C. It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA-dependent virulence factors in the PrfA-activating cytosolic compartment.

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Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependentplcA promoter. We have recently characterizedprfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcApromoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.

We recently reported the rapid expansion of an HIV-1 subtype F cluster among men who have sex with men (MSM) in the region of Galicia, Northwest Spain. Here we update this outbreak, analyze near full-length genomes, determine phylogenetic relationships, and estimate its origin. For this study, we used sequences of HIV-1 protease-reverse transcriptase and env V3 region, and for 17 samples, near full-length genome sequences were obtained. Phylogenetic analyses were performed via maximum likelihood. Locations and times of most recent common ancestors were estimated using Bayesian inference. Among samples analyzed by us, 100 HIV-1 F1 subsubtype infections of monophyletic origin were diagnosed in Spain, including 88 in Galicia and 12 in four other regions. Most viruses (n = 90) grouped in a subcluster (Galician subcluster), while 7 from Valladolid (Central Spain) grouped in another subcluster. At least 94 individuals were sexually-infected males and at least 71 were MSM. Seventeen near full-length genomes were uniformly of F1 subsubtype. Through similarity searches and phylogenetic analyses, we identified 18 viruses from four other Western European countries [Switzerland (n = 8), Belgium (n = 5), France (n = 3), and United Kingdom (n = 2)] and one from Brazil, from samples collected in 2005–2011, which branched within the subtype F cluster, outside of both Spanish subclusters, most of them corresponding to recently infected individuals. The most probable geographic origin and age of the Galician subcluster was Ferrol, Northwest Galicia, around 2007, while the Western European cluster probably emerged in Switzerland around 2002. In conclusion, a recently expanded HIV-1 subtype F cluster, the largest non-subtype B cluster reported in Western Europe, continues to spread among MSM in Spain; this cluster is part of a larger cluster with a wide geographic circulation in diverse Western European countries.