Update on emerging multidrug-resistant Rhodococcus equi.

Jose Vázquez-Boland, Jorge Val-Calvo and Mariela Scortti present a brief summary of the main aspects surrounding the recently identified multidrug-resistant Rhodococcus equi that emerged in the USA and the actions being taken to tackle the problem with support from the UK's Horserace Betting Levy Board.

Listeria InlB Expedites Vacuole Escape and Intracellular Proliferation by Promoting Rab7 Recruitment via Vps34.

Rapid phagosomal escape mediated by listeriolysin O (LLO) is a prerequisite for Listeria monocytogenes intracellular replication and pathogenesis. Escape takes place within minutes after internalization from vacuoles that are negative to the early endosomal Rab5 GTPase and positive to the late endosomal Rab7. Using mutant analysis, we found that the listerial invasin InlB was required for optimal intracellular proliferation of L. monocytogenes. Starting from this observation, we determined in HeLa cells that InlB promotes early phagosomal escape and efficient Rab7 acquisition by the Listeria-containing vacuole (LCV). Recruitment of the class III phosphoinositide 3-kinase (PI3K) Vps34 to the LCV and accumulation of its lipid product, phosphatidylinositol 3-phosphate (PI3P), two key endosomal maturation mediators, were also dependent on InlB. Small interfering RNA (siRNA) knockdown experiments showed that Vps34 was required for Rab7 recruitment and early (LLO-mediated) escape and supported InlB-dependent intracellular proliferation. Together, our data indicate that InlB accelerates LCV conversion into an escape-favorable Rab7 late phagosome via subversion of class III PI3K/Vps34 signaling. Our findings uncover a new function for the InlB invasin in Listeria pathogenesis as an intracellular proliferation-promoting virulence factor. IMPORTANCE Avoidance of lysosomal killing by manipulation of the endosomal compartment is a virulence mechanism assumed to be largely restricted to intravacuolar intracellular pathogens. Our findings are important because they show that cytosolic pathogens like L. monocytogenes, which rapidly escape the phagosome after internalization, can also extensively subvert endocytic trafficking as part of their survival strategy. They also clarify that, instead of delaying phagosome maturation (to allow time for LLO-dependent disruption, as currently thought), via InlB L. monocytogenes appears to facilitate the rapid conversion of the phagocytic vacuole into an escape-conducive late phagosome. Our data highlight the multifunctionality of bacterial virulence factors. At the cell surface, the InlB invasin induces receptor-mediated phagocytosis via class I PI3K activation, whereas after internalization it exploits class III PI3K (Vsp34) to promote intracellular survival. Systematically elucidating the mechanisms by which Listeria interferes with PI3K signaling all along the endocytic pathway may lead to novel anti-infective therapies.

International Spread of Multidrug-Resistant Rhodococcus equi.

A multidrug-resistant clone of the animal and human pathogen Rhodococcus equi, MDR-RE 2287, has been circulating among equine farms in the United States since the 2000s. We report the detection of MDR-RE 2287 outside the United States. Our finding highlights the risk for MDR-RE spreading internationally with horse movements.

Antimicrobial Resistance Spectrum Conferred by pRErm46 of Emerging Macrolide (Multidrug)-Resistant Rhodococcus equi.

Clonal multidrug resistance recently emerged in Rhodococcus equi, complicating the therapeutic management of this difficult-to-treat animal- and human-pathogenic actinomycete. The currently spreading multidrug-resistant (MDR) "2287" clone arose in equine farms upon acquisition, and coselection by mass macrolide-rifampin therapy, of the pRErm46 plasmid carrying the erm(46) macrolide-lincosamide-streptogramin resistance determinant, and of an rpoBS531F mutation. Here, we screened a collection of susceptible and macrolide-resistant R. equi strains from equine clinical cases using a panel of 15 antimicrobials against rapidly growing mycobacteria (RGM) and nocardiae and other aerobic actinomycetes (NAA). R. equi isolates-including MDR ones-were generally susceptible to linezolid, minocycline, tigecycline, amikacin, and tobramycin according to Staphylococcus aureus interpretive criteria, plus imipenem, cefoxitin, and ceftriaxone based on Clinical and Laboratory Standards Institute (CLSI) guidelines for RGM/NAA. Susceptibility to ciprofloxacin and moxifloxacin was borderline according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Molecular analyses linked pRErm46 to significantly increased MICs for trimethoprim-sulfamethoxazole and doxycycline, in addition to clarithromycin, within the RGM/NAA panel, and to streptomycin, spectinomycin, and tetracycline resistance. pRErm46 variants with spontaneous deletions in the class 1 integron (C1I) region, observed in ≈30% of erm(46)-positive isolates, indicated that the newly identified resistances were attributable to the C1I's sulfonamide (sul1) and aminoglycoside (aaA9) resistance cassettes and adjacent tetRA(33) determinant. Most MDR isolates carried the rpoBS531F mutation of the 2287 clone, while different rpoB mutations (S531L, S531Y) detected in two cases suggest the emergence of novel MDR R. equi strains.

Why Are Some Listeria monocytogenes Genotypes More Likely To Cause Invasive (Brain, Placental) Infection?

Although all isolates of the foodborne pathogen Listeria monocytogenes are considered to be pathogenic, epidemiological evidence indicates that certain serovar 4b lineages are more likely to cause severe invasive (neuromeningeal, maternal-fetal) listeriosis. Recently described as L. monocytogenes "hypervirulent" clones, no distinctive bacterial trait has been identified so far that could account for the differential pathogenicity of these strains. Here, we discuss some preliminary observations in experimentally infected mice suggesting that serovar 4b hypervirulent strains may have a hitherto unrecognized capacity for prolonged in vivo survival. We propose the hypothesis that protracted survivability in primary infection foci in liver and spleen-the first target organs after intestinal translocation-may cause L. monocytogenes serovar 4b hypervirulent clones to have a higher probability of secondary dissemination to brain and placenta.

Conservation of Rhodococcus equi (Magnusson 1923) Goodfellow and Alderson 1977 and rejection of Rhodococcus hoagii (Morse 1912) Kämpfer et al. 2014.

A recent taxonomic study confirmed the synonymy of Rhodococcus equi (Magnusson 1923) Goodfellow and Alderson 1977 and Corynebacterium hoagii (Morse 1912) Eberson 1918. As a result, both R. equi and C. hoagii were reclassified as Rhodococcus hoagii comb. nov. in application of the principle of priority of the Prokaryotic Code. Because R. equi is a well-known animal and zoonotic human pathogen, and a bacterial name solidly established in the veterinary and medical literature, we and others argued that the nomenclatural change may cause error and confusion and be potentially perilous. We have now additionally found that the nomenclatural type of the basonym C. hoagii, ATCC 7005T, does not correspond with the original description of the species C. hoagii in the early literature. Its inclusion as the C. hoagii type on the Approved Lists 1980 results in a change in the characters of the taxon and in C. hoagii designating two different bacteria. Moreover, ATCC 7005, the only strain in circulation under the name C. hoagii, does not have a well documented history; it is unclear why it was deposited as C. hoagii and a possible mix-up with a Corynebacterium (Rhodococcus) equi isolate is a reasonable assumption. We therefore request the rejection of Rhodococcus hoagii as a nomen ambiguum, nomen dubium and nomen perplexum in addition to nomen periculosum, and conservation of the name Rhodococcus equi, according to Rules 56ab of the Code.

Clonal Confinement of a Highly Mobile Resistance Element Driven by Combination Therapy in Rhodococcus equi.

Antibiotic use has been linked to changes in the population structure of human pathogens and the clonal expansion of multidrug-resistant (MDR) strains among healthcare- and community-acquired infections. Here we present a compelling example in a veterinary pathogen, Rhodococcus equi, the causative agent of a severe pulmonary infection affecting foals worldwide. We show that the erm(46) gene responsible for emerging macrolide resistance among equine R. equi isolates in the United States is part of a 6.9-kb transposable element, TnRErm46, actively mobilized by an IS481 family transposase. TnRErm46 is carried on an 87-kb conjugative plasmid, pRErm46, transferable between R. equi strains at frequencies up to 10-3 The erm(46) gene becomes stabilized in R. equi by pRErm46's apparent fitness neutrality and wholesale TnRErm46 transposition onto the host genome. This includes the conjugally exchangeable pVAPA virulence plasmid, enabling the possibility of cotransfer of two essential traits for survival in macrolide-treated foals in a single mating event. Despite its high horizontal transfer potential, phylogenomic analyses show that erm(46) is paradoxically confined to a specific R. equi clone, 2287. R. equi 2287 also carries a unique rpoBS531F mutation conferring high-level resistance to rifampin, systematically administered together with macrolides against rhodococcal pneumonia on equine farms. Our data illustrate that under sustained combination therapy, several independent "founder" genetic events are concurrently required for resistance, limiting not only its emergence but also, crucially, horizontal spread, ultimately determining multiresistance clonality.IMPORTANCE MDR clades arise upon acquisition of resistance traits, but the determinants of their clonal expansion remain largely undefined. Taking advantage of the unique features of Rhodococcus equi infection control in equine farms, involving the same dual antibiotic treatment since the 1980s (a macrolide and rifampin), this study sheds light into the determinants of multiresistance clonality and the importance of combination therapy in limiting the dissemination of mobile resistance elements. Clinically effective therapeutic alternatives against R. equi foal pneumonia are currently lacking, and the identified macrolide-rifampin MDR clone 2287 has serious implications. Still at early stages of evolution and local spread, R. equi 2287 may disseminate globally, posing a significant threat to the equine industry and, also, public health due to the risk of zoonotic transmission. The characterization of the 2287 clone and its resistance determinants will enable targeted surveillance and control interventions to tackle the emergence of MDR R. equi.

Control of Bacterial Virulence through the Peptide Signature of the Habitat.

To optimize fitness, pathogens selectively activate their virulence program upon host entry. Here, we report that the facultative intracellular bacterium Listeria monocytogenes exploits exogenous oligopeptides, a ubiquitous organic N source, to sense the environment and control the activity of its virulence transcriptional activator, PrfA. Using a genetic screen in adsorbent-treated (PrfA-inducing) medium, we found that PrfA is functionally regulated by the balance between activating and inhibitory nutritional peptides scavenged via the Opp transport system. Activating peptides provide essential cysteine precursor for the PrfA-inducing cofactor glutathione (GSH). Non-cysteine-containing peptides cause promiscuous PrfA inhibition. Biophysical and co-crystallization studies reveal that peptides inhibit PrfA through steric blockade of the GSH binding site, a regulation mechanism directly linking bacterial virulence and metabolism. L. monocytogenes mutant analysis in macrophages and our functional data support a model in which changes in the balance of antagonistic Opp-imported oligopeptides promote PrfA induction intracellularly and PrfA repression outside the host.

Correction: Epistatic control of intrinsic resistance by virulence genes in Listeria.

[This corrects the article DOI: 10.1371/journal.pgen.1007525.].

Epistatic control of intrinsic resistance by virulence genes in Listeria.

Elucidating the relationships between antimicrobial resistance and virulence is key to understanding the evolution and population dynamics of resistant pathogens. Here, we show that the susceptibility of the gram-positive bacterium Listeria monocytogenes to the antibiotic fosfomycin is a complex trait involving interactions between resistance and virulence genes and the environment. We found that a FosX enzyme encoded in the listerial core genome confers intrinsic fosfomycin resistance to both pathogenic and non-pathogenic Listeria spp. However, in the genomic context of the pathogenic L. monocytogenes, FosX-mediated resistance is epistatically suppressed by two members of the PrfA virulence regulon, hpt and prfA, which upon activation by host signals induce increased fosfomycin influx into the bacterial cell. Consequently, in infection conditions, most L. monocytogenes isolates become susceptible to fosfomycin despite possessing a gene that confers high-level resistance to the drug. Our study establishes the molecular basis of an epistatic interaction between virulence and resistance genes controlling bacterial susceptibility to an antibiotic. The reported findings provide the rationale for the introduction of fosfomycin in the treatment of Listeria infections even though these bacteria are intrinsically resistant to the antibiotic in vitro.

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes.

The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA-/LLO-) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA-/LLO- mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.

Listeria Placental Infection.

The Gram-positive facultative intracellular bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection. Pregnant women are at risk of contracting listeriosis, which can potentially lead to miscarriage, stillbirth, preterm birth, and congenital neonatal infections. While other systemic bacterial infections may result in adverse pregnancy outcomes at comparable frequencies, L. monocytogenes has particular notoriety because fetal complications largely occur in the absence of overt illness in the mother, delaying medical intervention. Here, we briefly review the pathophysiology and mechanisms of maternofetal listeriosis, discussed in light of a recent mBio report on Listeria transplacental infection in a nonhuman primate model.

Comparative Genomics of Rhodococcus equi Virulence Plasmids Indicates Host-Driven Evolution of the vap Pathogenicity Island.

The conjugative virulence plasmid is a key component of the Rhodococcus equi accessory genome essential for pathogenesis. Three host-associated virulence plasmid types have been identified the equine pVAPA and porcine pVAPB circular variants, and the linear pVAPN found in bovine (ruminant) isolates. We recently characterized the R. equi pangenome (Anastasi E, et al. 2016. Pangenome and phylogenomic analysis of the pathogenic actinobacterium Rhodococcus equi. Genome Biol Evol. 8:3140-3148.) and we report here the comparative analysis of the virulence plasmid genomes. Plasmids within each host-associated type were highly similar despite their diverse origins. Variation was accounted for by scattered single nucleotide polymorphisms and short nucleotide indels, while larger indels-mostly in the plasticity region near the vap pathogencity island (PAI)-defined plasmid genomic subtypes. Only one of the plasmids analyzed, of pVAPN type, was exceptionally divergent due to accumulation of indels in the housekeeping backbone. Each host-associated plasmid type carried a unique PAI differing in vap gene complement, suggesting animal host-specific evolution of the vap multigene family. Complete conservation of the vap PAI was observed within each host-associated plasmid type. Both diversity of host-associated plasmid types and clonality of specific chromosomal-plasmid genomic type combinations were observed within the same R. equi phylogenomic subclade. Our data indicate that the overall strong conservation of the R. equi host-associated virulence plasmids is the combined result of host-driven selection, lateral transfer between strains, and geographical spread due to international livestock exchanges.

Pangenome and Phylogenomic Analysis of the Pathogenic Actinobacterium Rhodococcus equi.

We report a comparative study of 29 representative genomes of the animal pathogen Rhodococcus equi The analyses showed that R. equi is genetically homogeneous and clonal, with a large core genome accounting for ≈80% of an isolates' gene content. An open pangenome, even distribution of accessory genes among the isolates, and absence of significant core-genome recombination, indicated that gene gain/loss is a main driver of R. equi genome evolution. Traits previously predicted to be important in R. equi physiology, virulence and niche adaptation were part of the core genome. This included the lack of a phosphoenolpyruvate:carbohydrate transport system (PTS), unique among the rhodococci except for the closely related Rhodococcus defluvii, reflecting selective PTS gene loss in the R. equi-R. defluvii sublineage. Thought to be asaccharolytic, rbsCB and glcP non-PTS sugar permease homologues were identified in the core genome and, albeit inefficiently, R. equi utilized their putative substrates, ribose and (irregularly) glucose. There was no correlation between R. equi whole-genome phylogeny and host or geographical source, with evidence of global spread of genomovars. The distribution of host-associated virulence plasmid types was consistent with the exchange of the plasmids (and corresponding host shifts) across the R. equi population, and human infection being zoonotically acquired. Phylogenomic analyses demonstrated that R. equi occupies a central position in the Rhodococcus phylogeny, not supporting the recently proposed transfer of the species to a new genus.

PrfA regulation offsets the cost of Listeria virulence outside the host.

Virulence traits are essential for pathogen fitness, but whether they affect microbial performance in the environment, where they are not needed, remains experimentally unconfirmed. We investigated this question with the facultative pathogen Listeria monocytogenes and its PrfA virulence regulon. PrfA-regulated genes are activated intracellularly (PrfA 'ON') but shut down outside the host (PrfA 'OFF'). Using a mutant PrfA regulator locked ON (PrfA*) and thus causing PrfA-controlled genes to be constitutively activated, we show that virulence gene expression significantly impairs the listerial growth rate (µ) and maximum growth (A) in rich medium. Deletion analysis of the PrfA regulon and complementation of a L. monocytogenes mutant lacking all PrfA-regulated genes with PrfA* indicated that the growth reduction was specifically due to the unneeded virulence determinants and not to pleiotropic regulatory effects of PrfA ON. No PrfA*-associated fitness disadvantage was observed in infected eukaryotic cells, where PrfA-regulated virulence gene expression is critical for survival. Microcosm experiments demonstrated that the constitutively virulent state strongly impaired L. monocytogenes performance in soil, the natural habitat of these bacteria. Our findings provide empirical proof that virulence carries a significant cost to the pathogen. They also experimentally substantiate the assumed, although not proven, key role of virulence gene regulation systems in suppressing the cost of bacterial virulence outside the host.

An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.

We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species.

Mouse lung infection model to assess Rhodococcus equi virulence and vaccine protection.

The pathogenic actinomycete Rhodococcus equi causes severe purulent lung infections in foals and immunocompromised people. Although relatively unsusceptible to R. equi, mice are widely used for in vivo studies with this pathogen. The most commonly employed mouse model is based on systemic (intravenous) infection and determination of R. equi burdens in spleen and liver. Here, we investigated the murine lung for experimental infection studies with R. equi. Using a 10(7)CFU intranasal challenge in BALB/c mice, virulent R. equi consistently survived in quantifiable numbers up to 10 days in the lungs whereas virulence-deficient R. equi bacteria were rapidly cleared. An internally controlled virulence assay was developed in which the test R. equi strains are co-inoculated and monitored in the same mouse. Isogenic R. equi bacteria lacking either the plasmid vapA gene or the entire virulence plasmid were compared using this competitive assay. Both strains showed no significant differences in in vivo fitness in the lung, indicating that the single loss of the virulence factor VapA was sufficient to account for the full attenuation seen in the absence of the virulence plasmid. To test the adequacy of the lung infection model for monitoring R. equi vaccine efficacy, BALB/c mice were immunized with live R. equi and challenged intranasally. Vaccination conferred protection against acute pulmonary challenge with virulent R. equi. Our data indicate that the murine lung infection model provides a useful tool for both R. equi virulence and vaccine studies.

Comparison of Listeria monocytogenes Exoproteomes from biofilm and planktonic state: Lmo2504, a protein associated with biofilms.

The food-borne pathogen Listeria monocytogenes is the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119Δlmo2504), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119Δlmo2504 showed a significantly (P < 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities.

Genome and proteome analysis of phage E3 infecting the soil-borne actinomycete Rhodococcus equi.

We report on the characterization and genomic analysis of bacteriophage E3 isolated from soil and propagating in Rhodococcus equi strains. Phage E3 has a circular genome of 142 563 bp and is the first Myoviridae reported for the genus Rhodococcus and for a non-mycobacterial actinomycete. Phylogenetic analyses placed E3 in a distinct Myoviridae clade together with Mycobacterium phages Bxz1 and Myrna. The highly syntenic genomes of this myoviridal group comprise vertically evolving core phage modules flanked by hyperplastic regions specific to each phage and rich in horizontally acquired DNA. The hyperplastic regions contain numerous tRNA genes in the mycobacteriophages which are absent in E3, possibly reflecting bacterial host-specific translation-related phage fitness constraints associated with rate-limiting tRNAs. A structural proteome analysis identified 28 E3 polypeptides, including 15 not previously known to be virion-associated proteins. The E3 genome and comparative analysis provide insight into short-term genome evolution and adaptive plasticity in tailed phages from the environmental microbiome.

Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo.

The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes. PrfA-dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand-induced allostery. Here, we report the identification of a solvent-accessible cavity within the PrfA N-terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide-anchoring motif and has its entrance on the opposite side of the ß-barrel. Site-directed mutations in this pocket impaired intracellular PrfA-dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation-deficient PrfA mutations. Neither PrfA(allo) substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell-to-cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues.