An Actinobacillus pleuropneumoniae arcA deletion mutant is attenuated and deficient in biofilm formation

Buettner, Falk F. R.; Maas, Alexander; Gerlach, Gerald-F.

doi:10.1016/j.vetmic.2007.08.005

Cited by 47 publications

(33 citation statements)

References 28 publications

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“…Potential regulatory proteins and cues that affect biofilm formation in A. pleuropneumoniae have been identified. Buettner et al reported that an A. pleuropneumoniae serotype 7 arcA mutant strain was defective in autoaggregation and biofilm formation on glass surfaces, suggesting that ArcA positively regulates biofilm formation (34). ArcAB is a two-component global regulatory system involved in the adaptation to growth under anaerobic conditions and is required for full virulence of A. pleuropneumoniae and for persistence in chronic infection (34).…”

Section: Discussionmentioning

confidence: 99%

“…Buettner et al reported that an A. pleuropneumoniae serotype 7 arcA mutant strain was defective in autoaggregation and biofilm formation on glass surfaces, suggesting that ArcA positively regulates biofilm formation (34). ArcAB is a two-component global regulatory system involved in the adaptation to growth under anaerobic conditions and is required for full virulence of A. pleuropneumoniae and for persistence in chronic infection (34). A luxS mutant strain of A. pleuropneumoniae serotype 1, defective in the biosynthesis of quorum-sensing signal autoinducer 2, was shown to produce more biofilm than the parent strain (35).…”

Section: Discussionmentioning

confidence: 99%

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The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

et al. 2013

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Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-␤-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 ؋ 10 ؊5). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

et al. 2013

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“…For RNA and protein preparations, this medium was inoculated with 1% of an aerobically grown log-phase culture in supplemented PPLO medium with an optical density at 600 nm (OD 600 ) of 0.3, and bacteria were grown anaerobically for 6 h, until they reached late exponential growth phase, and were then harvested by centrifugation. Due to severe autoaggregation under anaerobic conditions, the growth phase was assessed by determination of the total protein content (8).…”

Section: Methodsmentioning

confidence: 99%

“…Comparisons of the virulence of the A. pleuropneumoniae wt and the frd deletion mutant were assessed by using an aerosol infection model after determining clinical, lung, and reisolation scores as previously described (8). Briefly, A. pleuropneumoniae-free pigs (German Landrace, male, 8 to 9 weeks of age) were randomly assigned to two groups (wt group, 7 pigs; ⌬frd group, 8 Based on the specifications for the validation of A. pleuropneumoniae vaccines given in the European Pharmacopoeia (http://www.pheur.org), the occurrence of coughing, dyspnea, and vomiting was assessed daily within the first 7 days postinfection. In order to quantify the results, a score of 1 was given for each of the symptoms, resulting in a maximum score of 3 per pig and day.…”

Section: Methodsmentioning

confidence: 99%

Analysis of theActinobacillus pleuropneumoniaeArcA Regulon Identifies Fumarate Reductase as a Determinant of Virulence

Buettner¹,

Bendallah²,

Bossé

et al. 2008

Infect Immun

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The ability of the bacterial pathogen Actinobacillus pleuropneumoniae to grow anaerobically allows the bacterium to persist in the lung. The ArcAB two-component system is crucial for metabolic adaptation in response to anaerobic conditions, and we recently showed that an A. pleuropneumoniae arcA mutant had reduced virulence compared to the wild type (F. F. Buettner, A. Maas, and G.-F. Gerlach, Vet. Microbiol. 127:106-115, 2008). In order to understand the attenuated phenotype, we investigated the ArcA regulon of A. pleuropneumoniae by using a combination of transcriptome (microarray) and proteome (two-dimensional difference gel electrophoresis and subsequent mass spectrometry) analyses. We show that ArcA negatively regulates the expression of many genes, including those encoding enzymes which consume intermediates during fumarate synthesis. Simultaneously, the expression of glycerol-3-phosphate dehydrogenase, a component of the respiratory chain serving as a direct reduction equivalent for fumarate reductase, was upregulated. This result, together with the in silico analysis finding that A. pleuropneumoniae has no oxidative branch of the citric acid cycle, led to the hypothesis that fumarate reductase might be crucial for virulence by providing (i) energy via fumarate respiration and (ii) succinate and other essential metabolic intermediates via the reductive branch of the citric acid cycle. To test this hypothesis, an isogenic A. pleuropneumoniae fumarate reductase deletion mutant was constructed and studied by using a pig aerosol infection model. The mutant was shown to be significantly attenuated, thereby strongly supporting a crucial role for fumarate reductase in the pathogenesis of A. pleuropneumoniae infection.Actinobacillus pleuropneumoniae is the causative agent of a porcine pleuropneumonia that results in high economic losses worldwide (16). After A. pleuropneumoniae-containing aerosols are inhaled, the pathogen colonizes the respiratory epithelium of its host and persists on tonsils, on healthy lung epithelium, and in sequestered lesions (14). The persistence of bacterial pathogens in the respiratory tracts of clinically healthy carriers is of major importance in the spread of infection in both animals and humans (6,10,29).Previously, we have shown that the ability of A. pleuropneumoniae to adapt to low redox conditions is essential for its long-term persistence on intact and diseased respiratory tract epithelia (4, 26). In particular, the arcA deletion mutant of A. pleuropneumoniae was severely attenuated in this respect (8). A role in virulence for ArcA has also been implicated for intracellular bacterial pathogens such as Mycobacterium tuberculosis (42,45), invasive pathogens such as Haemophilus influenzae (13, 59), and the enteric pathogens Vibrio cholerae (50) and Shigella flexneri (7). However, the molecular mechanisms responsible for this attenuation are only partially resolved.In Escherichia coli, the transcriptional regulator ArcA functions primarily as a major metabolic modulator, downregula...

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“…A. pleuropneumoniae virulence is thought to be dependent on a variety of putative virulence determinants, including lipopolysaccharide (5,32), capsule (4), iron binding proteins (2,33), Apx toxins (17), type 4 fimbriae (7,40), and flagella (30). Furthermore, proteins involved with anaerobic metabolic activity appear to be required for persistence on porcine lung epithelia (3,10,24).…”

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confidence: 99%

Functional Characterization of AasP, a Maturation Protease Autotransporter Protein ofActinobacillus pleuropneumoniae

et al. 2008

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Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a highly contagious respiratory infection in pigs. AasP, a putative subtilisin-like serine protease autotransporter, has recently been identified in A. pleuropneumoniae. We hypothesized that, similarly to other autotransporters of this type, AasP may undergo autocatalytic cleavage resulting in release of the passenger domain of the protein. Furthermore, AasP may be responsible for cleavage of other A. pleuropneumoniae outer membrane proteins. To address these hypotheses, the aasP gene was cloned and the expressed recombinant AasP protein used to raise monospecific rabbit antiserum. Immunoblot analysis of whole-cell lysates and secreted proteins demonstrated that AasP does not undergo proteolytic cleavage. Immunoblot analysis also confirmed that AasP is universally expressed by A. pleuropneumoniae. Confirmation of the maturation protease function of AasP was obtained through phenotypic analysis of an A. pleuropneumoniae aasP deletion mutant and by functional complementation. Comparison of the secreted proteins of the wild type, an aasP mutant derivative, and an aasP mutant complemented in trans led to the identification of OmlA protein fragments that were present only in the secreted-protein preparations of the wild-type and complemented strains, indicating that AasP is involved in modification of OmlA. This is the first demonstration of a function for any autotransporter protein in Actinobacillus pleuropneumoniae.

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An Actinobacillus pleuropneumoniae arcA deletion mutant is attenuated and deficient in biofilm formation

Cited by 47 publications

References 28 publications

The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

Analysis of theActinobacillus pleuropneumoniaeArcA Regulon Identifies Fumarate Reductase as a Determinant of Virulence

Functional Characterization of AasP, a Maturation Protease Autotransporter Protein ofActinobacillus pleuropneumoniae

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