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

We report a novel host-associated virulence plasmid inRhodococcus 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. equiisolates. pVAPN is similar to the linear plasmid pNSL1 fromRhodococcussp. NS1 and harbors six newvapmultigene family members (vapNtovapS) in avappathogenicity locus presumably acquired viaen blocmobilization from a direct predecessor of equine pVAPA. Loss of pVAPN renderedR. equiavirulent in macrophages and mice. Mating experiments using anin vivotransconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-curedR. equirecipient. Phylogenetic analyses assigned thevapmultigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursorvapgene carried by the nearestvapisland ancestor. Deletion ofvapN, the predicted “bovine-type” allelic counterpart ofvapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in whichR. equivirulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a keyvapdeterminant present in the common ancestor of the plasmid-specificvapislands, with host tropism as a secondary trait selected during coevolution with specific animal species.

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Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages

mSphere ◽

10.1128/msphere.00186-16 ◽

2016 ◽

Vol 1 (5) ◽

Cited By ~ 6

Author(s):

Jennifer M. Willingham-Lane ◽

Londa J. Berghaus ◽

Steeve Giguère ◽

Mary K. Hondalus

Keyword(s):

Host Species ◽

Opportunistic Pathogen ◽

Rhodococcus Equi ◽

Replication Capacity ◽

Virulence Plasmid ◽

Intracellular Replication ◽

Content Type ◽

Disease Agent ◽

Species Specific

ABSTRACT This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a conjugative virulence plasmid, pVAPA1037, that expresses Vap proteins, including VapA, essential for intramacrophage replication and virulence in vivo. The understudied R. equi isolates from pigs carry a related but different plasmid, pVAPB, expressing distinct Vap proteins, including VapB. In this work, we document for the first time that R. equi isolates carrying pVAPB-type plasmids are capable of intramacrophage replication. Moreover, we show that R. equi isolates carrying either plasmid type can replicate in both equine and swine macrophages, indicating that host species tropism is not due to species-specific intramacrophage replication capabilities defined by plasmid type. Furthermore, plasmid swapping between equine and swine strains did not alter intracellular replication capacity, indicating that coevolution of the plasmid and chromosome is not essential for intracellular growth. The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a conjugative virulence plasmid, pVAPA1037, that expresses Vap proteins, including VapA, essential for intramacrophage replication and virulence in vivo. The understudied R. equi isolates from pigs carry a related but different plasmid, pVAPB, expressing distinct Vap proteins, including VapB. In this work, we document for the first time that R. equi isolates carrying pVAPB-type plasmids are capable of intramacrophage replication. Moreover, we show that R. equi isolates carrying either plasmid type can replicate in both equine and swine macrophages, indicating that host species tropism is not due to species-specific intramacrophage replication capabilities defined by plasmid type. Furthermore, plasmid swapping between equine and swine strains did not alter intracellular replication capacity, indicating that coevolution of the plasmid and chromosome is not essential for intracellular growth.

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Persistence Dynamics of Antimicrobial-Resistant Neisseria in the Pharynx of Rhesus Macaques

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02232-19 ◽

2020 ◽

Vol 64 (8) ◽

Author(s):

Eliza Thapa ◽

Hanna M. Knauss ◽

Benjamin A. Colvin ◽

Benjamin A. Fischer ◽

Nathan J. Weyand

Keyword(s):

Rhesus Macaques ◽

Fluoroquinolone Resistance ◽

Host Animal ◽

Content Type ◽

Host Tropism ◽

Animal Modeling ◽

Neisseria Mucosa ◽

Previously Treated

ABSTRACT Pharyngeal infections by Neisseria gonorrhoeae are often asymptomatic, making them difficult to treat. However, in vivo animal modeling of human pharyngeal infections by pathogenic Neisseria species is challenging due to numerous host tropism barriers. We have relied on rhesus macaques to investigate pharyngeal persistence of naturally occurring Neisseria species in response to antibiotics. These species include Neisseria mucosa, Neisseria oralis, and a species unique to macaques. Four animals previously treated intramuscularly with the fluoroquinolone enrofloxacin for 2 weeks were monitored for persistence of their preexisting Neisseria populations for a period of 10 weeks. Enrofloxacin exposure did not eliminate preexisting flora from two of the four animals. Characterization of a collection of macaque Neisseria isolates supported the hypothesis that pharyngeal persistence was linked to reduced enrofloxacin susceptibility conferred by mutations in either gyrA or parC. Interestingly, we observed a change in neisserial population dynamics for several weeks following enrofloxacin exposure. Enrofloxacin appeared to promote competition between strains for dominance in the pharyngeal niche. Specifically, following enrofloxacin treatment, strains bearing single gyrA mutations and low MICs persisted long-term. In contrast, strains with both gyrA and parC mutations and high MICs became culturally undetectable, consistent with the hypothesis that they were less fit. Our study has provided insight into pharyngeal persistence dynamics of Neisseria species bearing fluoroquinolone resistance determinants. The rhesus macaque provides a valuable host animal that may be used in the future to simulate treatment failures associated with the presence of antimicrobial-resistant Neisseria spp. in the human pharynx.

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Characterization of the Role of the Pathogenicity Island and vapG in the Virulence of the Intracellular Actinomycete Pathogen Rhodococcus equi

Infection and Immunity ◽

10.1128/iai.00081-10 ◽

2010 ◽

Vol 78 (8) ◽

pp. 3323-3334 ◽

Cited By ~ 27

Author(s):

Garry B. Coulson ◽

Shruti Agarwal ◽

Mary K. Hondalus

Keyword(s):

Deletion Mutant ◽

Virulence Genes ◽

Pathogenicity Island ◽

Rhodococcus Equi ◽

Virulence Plasmid ◽

Immune Deficiencies ◽

Life Threatening ◽

Positive Regulators ◽

Important Virulence Factor

ABSTRACT Rhodococcus equi, a facultative intracellular pathogen of macrophages, causes severe, life-threatening pneumonia in young foals and in people with underlying immune deficiencies. R. equi virulence is dependent on the presence of a large virulence plasmid that houses a pathogenicity island (PAI) encoding a novel family of surface-localized and secreted proteins of largely unknown function termed the virulence-associated proteins (VapACDEFGHI). To date, vapA and its positive regulators virR and orf8 are the only experimentally established virulence genes residing on the virulence plasmid. In this study, a PAI deletion mutant was constructed and, as anticipated, was attenuated for growth both in macrophages and in mice due to the absence of vapA expression. Expression of vapA in the PAI mutant from a constitutive promoter, thereby eliminating the requirement for the PAI-encoded vapA regulators, resulted in delayed bacterial clearance in vivo, yet full virulence was not restored, indicating that additional virulence genes are indeed located within the deleted pathogenicity island region. Based on previous reports demonstrating that the PAI-carried gene vapG is highly upregulated in macrophages and in the lungs of R. equi-infected foals, we hypothesized that vapG could be an important virulence factor. However, analysis of a marked vapG deletion mutant determined the gene to be dispensable for growth in macrophages and in vivo in mice.

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Phosphinothricin Acetyltransferases Identified UsingIn Vivo,In Vitro, and Bioinformatic Analyses

Applied and Environmental Microbiology ◽

10.1128/aem.02604-16 ◽

2016 ◽

Vol 82 (24) ◽

pp. 7041-7051 ◽

Cited By ~ 5

Author(s):

Chelsey M. VanDrisse ◽

Kristy L. Hentchel ◽

Jorge C. Escalante-Semerena

Keyword(s):

Deinococcus Radiodurans ◽

Phylogenetic Analyses ◽

Critical Role ◽

Metabolic Stress ◽

Physiological Role ◽

Methionine Sulfoximine ◽

Acinetobacter Baylyi ◽

Content Type

ABSTRACTAcetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals.Streptomycesspecies utilize aGcn5N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin,phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA fromSalmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases.S. entericawas used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO.In vitroandin vivoanalyses identified substrates acetylated by putative PPT acetyltransferases fromDeinococcus radiodurans(DR_1057 and DR_1182) andGeobacillus kaustophilus(GK0593 and GK2920).In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results ofin vitrostudies were consistent with thein vivoresults. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases inBurkholderia xenovorans,Bacillus subtilis,Staphylococcus aureus,Acinetobacter baylyi, andEscherichia coli.IMPORTANCEThe work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role ofGcn5N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination ofin vivo,in vitro, and bioinformatics approaches reported here identified GNATs that can acetylate and detoxify phosphinothricin.

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Success of Escherichia coli O25b:H4 Sequence Type 131 Clade C Associated with a Decrease in Virulence

Infection and Immunity ◽

10.1128/iai.00576-20 ◽

2020 ◽

Vol 88 (12) ◽

Author(s):

Marion Duprilot ◽

Alexandra Baron ◽

François Blanquart ◽

Sara Dion ◽

Cassandra Pouget ◽

...

Keyword(s):

Escherichia Coli ◽

Phylogenetic Analyses ◽

Multidrug Resistant ◽

Sequence Type ◽

Content Type ◽

Gut Colonization ◽

Clade B ◽

Clade C ◽

Severe Infections

ABSTRACT Escherichia coli O25b:H4 sequence type 131 (ST131), which is resistant to fluoroquinolones and which is a producer of CTX-M-15, is globally one of the major extraintestinal pathogenic E. coli (ExPEC) lineages. Phylogenetic analyses showed that multidrug-resistant ST131 strains belong to clade C, which recently emerged from clade B by stepwise evolution. It has been hypothesized that features other than multidrug resistance could contribute to this dissemination since other major global ExPEC lineages (ST73 and ST95) are mostly antibiotic susceptible. To test this hypothesis, we compared early biofilm production, presence of ExPEC virulence factors (VFs), and in vivo virulence in a mouse sepsis model in 19 and 20 epidemiologically relevant strains of clades B and C, respectively. Clade B strains were significantly earlier biofilm producers (P < 0.001), carriers of more VFs (P = 4e−07), and faster killers of mice (P = 2e−10) than clade C strains. Gene inactivation experiments showed that the H30-fimB and ibeART genes were associated with in vivo virulence. Competition assays in sepsis, gut colonization, and urinary tract infection models between the most anciently diverged strain (B1 subclade), one C1 subclade strain, and a B4 subclade recombining strain harboring some clade C-specific genetic events showed that the B1 strain always outcompeted the C1 strain, whereas the B4 strain outcompeted the C1 strain, depending on the mouse niches. All these findings strongly suggest that clade C evolution includes a progressive loss of virulence involving multiple genes, possibly enhancing overall strain fitness by avoiding severe infections, even if it comes at the cost of a lower colonization ability.

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

mBio ◽

10.1128/mbio.02260-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 7

Author(s):

Sonsiray Álvarez-Narváez ◽

Steeve Giguère ◽

Elisa Anastasi ◽

Jack Hearn ◽

Mariela Scortti ◽

...

Keyword(s):

Combination Therapy ◽

Antibiotic Use ◽

Clonal Expansion ◽

The United States ◽

Multidrug Resistant ◽

Rhodococcus Equi ◽

Conjugative Plasmid ◽

Virulence Plasmid ◽

Human Pathogens ◽

Content Type

ABSTRACT 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.

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Molecular and Clinical Characterization of Multidrug-Resistant and Hypervirulent Klebsiella pneumoniae Strains from Liver Abscess in Taiwan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00174-20 ◽

2020 ◽

Vol 64 (5) ◽

Cited By ~ 6

Author(s):

Yi-Tsung Lin ◽

Yi-Hsiang Cheng ◽

Chien Chuang ◽

Sheng-Hua Chou ◽

Wan-Hsin Liu ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Liver Abscess ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Virulence Plasmid ◽

Content Type ◽

Sequence Types

ABSTRACT Hypervirulent Klebsiella pneumoniae strains are the major cause of liver abscesses throughout East Asia, and these strains are usually antibiotic susceptible. Recently, multidrug-resistant and hypervirulent (MDR-HV) K. pneumoniae strains have emerged due to hypervirulent strains acquiring antimicrobial resistance determinants or the transfer of a virulence plasmid into a classic MDR strain. In this study, we characterized the clinical and microbiological properties of K. pneumoniae liver abscess (KPLA) caused by MDR-HV strains in Taiwan. Patients with community onset KPLA were retrospectively identified at Taipei Veterans General Hospital during January 2013 to May 2018. Antimicrobial resistance mechanisms, capsular types, and sequence types were determined. MDR-HV strains and their parental antimicrobial-susceptible strains further underwent whole-genome sequencing (WGS) and in vivo mice lethality tests. Thirteen MDR-HV strains were identified from a total of 218 KPLA episodes. MDR-HV strains resulted in similar outcomes to antimicrobial-susceptible strains. All MDR-HV strains were traditional hypervirulent clones carrying virulence capsular types. The major resistance mechanisms were the overexpression of efflux pumps and/or the acquisition of ESBL or AmpC β-lactamase genes. WGS revealed that two hypervirulent strains had evolved to an MDR phenotype due to mutation in the ramR gene and the acquisition of an SHV-12-bearing plasmid, respectively. Both these MDR-HV strains retained high virulence compared to their parental strains. The spread of MDR-HV K. pneumoniae strains in the community raises significant public concerns, and measures should be taken to prevent the further acquisition of carbapenemase and other resistance genes among these strains in order to avoid the occurrence of untreatable KPLA.

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Effect of Macrolide and Rifampin Resistance on Fitness of Rhodococcus equi during Intramacrophage Replication and In Vivo

Infection and Immunity ◽

10.1128/iai.00281-19 ◽

2019 ◽

Vol 87 (10) ◽

Cited By ~ 2

Author(s):

Jennifer M. Willingham-Lane ◽

Londa J. Berghaus ◽

Roy D. Berghaus ◽

Kelsey A. Hart ◽

Steeve Giguère

Keyword(s):

Resistant Strain ◽

Clinical Signs ◽

Rhodococcus Equi ◽

Infection Model ◽

Content Type ◽

Mouse Infection Model ◽

Pulmonary Macrophages ◽

Rifampin Resistance

ABSTRACT The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a facultative intracellular pathogen of macrophages and causes severe bronchopneumonia when inhaled by susceptible foals. Standard treatment for R. equi disease is dual-antimicrobial therapy with a macrolide and rifampin. Thoracic ultrasonography and early treatment with antimicrobials prior to the development of clinical signs are used as means of controlling endemic R. equi infection on many farms. Concurrently with the increased use of macrolides and rifampin for chemoprophylaxis and the treatment of subclinically affected foals, a significant increase in the incidence of macrolide- and rifampin-resistant R. equi isolates has been documented. Previously, our laboratory demonstrated decreased fitness of R. equi strains that were resistant to macrolides, rifampin, or both, resulting in impaired in vitro growth in iron-restricted media and in soil. The objective of this study was to examine the effect of macrolide and/or rifampin resistance on intracellular replication of R. equi in equine pulmonary macrophages and in an in vivo mouse infection model in the presence and absence of antibiotics. In equine macrophages, the macrolide-resistant strain did not increase in bacterial numbers over time and the dual macrolide- and rifampin-resistant strain exhibited decreased proliferation compared to the susceptible isolate. In the mouse model, in the absence of antibiotics, the susceptible R. equi isolate outcompeted the macrolide- or rifampin-resistant strains.

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The Hydroxamate Siderophore Rhequichelin Is Required for Virulence of the Pathogenic Actinomycete Rhodococcus equi

Infection and Immunity ◽

10.1128/iai.00678-12 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4106-4114 ◽

Cited By ~ 21

Author(s):

Raúl Miranda-CasoLuengo ◽

Garry B. Coulson ◽

Aleksandra Miranda-CasoLuengo ◽

José A. Vázquez-Boland ◽

Mary K. Hondalus ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Iron Acquisition ◽

Iron Chelator ◽

Rhodococcus Equi ◽

Wild Type ◽

Content Type ◽

Peptide Synthetase ◽

Tailoring Enzymes

ABSTRACTWe previously showed that the facultative intracellular pathogenRhodococcus equiproduces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that therhbABCDEcluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. TherhbCgene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting ofN5-formyl-N5-hydroxyornithine, serine,N5-hydroxyornithine, andN5-acyl-N5-hydroxyornithine. The otherrhbgenes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription ofrhbCwas upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion ofrhbCD, rendering the resulting strainR. equiSID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype ofR. equiSID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels ofrhbCfollowing infection and the inability ofR. equiSID2 to grow within macrophages. Unlike the wild-type strain,R. equiSID2 was unable to replicatein vivoand was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenicRhodococcusspecies also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution ofR. equivirulence.

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Genome Expression Analysis of Nonproliferating Intracellular Salmonella enterica Serovar Typhimurium Unravels an Acid pH-Dependent PhoP-PhoQ Response Essential for Dormancy

Infection and Immunity ◽

10.1128/iai.01080-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 154-165 ◽

Cited By ~ 31

Author(s):

Cristina Núñez-Hernández ◽

Alberto Tierrez ◽

Álvaro D. Ortega ◽

M. Graciela Pucciarelli ◽

Marta Godoy ◽

...

Keyword(s):

Metabolic Reprogramming ◽

Host Cells ◽

Infection Model ◽

Intracellular Bacteria ◽

Virulence Plasmid ◽

Intracellular Growth ◽

Content Type ◽

Serovar Typhimurium

Genome-wide expression analyses have provided clues on howSalmonellaproliferates inside cultured macrophages and epithelial cells. However,in vivostudies show thatSalmonelladoes not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined.In vitroinfection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurringin vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in whichS. entericaserovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model thatS. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of theS. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate thatS. Typhimurium restrains intracellular growthin vivoand support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.

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