Membrane morphology and leukotoxin secretion are associated with a novel membrane protein of Aggregatibacter actinomycetemcomitans.

Gram-negative bacteria display either a flat or an irregular outer membrane. The periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans has an irregular outer membrane. We have identified a gene that is associated with the biogenesis of this morphology. The gene is part of a three-gene operon and codes for a 141-kDa protein designated morphogenesis protein C (MorC), which is conserved in several gram-negative bacteria including Haemophilus influenzae and Pasteurella multocida. Insertional inactivation of this gene resulted in the conversion of an irregularly shaped membrane to a flat membrane. Associated with this morphological change were the autoaggregation of the bacteria during planktonic growth and a concomitant increase in the surface hydrophobicity of the bacterium. The absence of MorC also resulted in the loss of the secretion of leukotoxin but not the ltxA transcription. Our findings suggest that MorC is critical for membrane morphology and leukotoxin secretion in A. actinomycetemcomitans.

Common beta-lactamases inhibit bacterial biofilm formation.

Beta-lactamases, which evolved from bacterial penicillin-binding proteins (PBPs) involved in peptidoglycan (PG) synthesis, confer resistance to beta-lactam antibiotics. While investigating the genetic basis of biofilm development by Pseudomonas aeruginosa, we noted that plasmid vectors encoding the common beta-lactamase marker TEM-1 caused defects in twitching motility (mediated by type IV pili), adherence and biofilm formation without affecting growth rates. Similarly, strains of Escherichia coli carrying TEM-1-encoding vectors grew normally but showed reduced adherence and biofilm formation, showing this effect was not species-specific. Introduction of otherwise identical plasmid vectors carrying tetracycline or gentamicin resistance markers had no effect on biofilm formation or twitching motility. The effect is restricted to class A and D enzymes, because expression of the class D Oxa-3 beta-lactamase, but not class B or C beta-lactamases, impaired biofilm formation by E. coli and P. aeruginosa. Site-directed mutagenesis of the catalytic Ser of TEM-1, but not Oxa-3, abolished the biofilm defect, while disruption of either TEM-1 or Oxa-3 expression restored wild-type levels of biofilm formation. We hypothesized that the A and D classes of beta-lactamases, which are related to low molecular weight (LMW) PBPs, may sequester or alter the PG substrates of such enzymes and interfere with normal cell wall turnover. In support of this hypothesis, deletion of the E. coli LMW PBPs 4, 5 and 7 or combinations thereof, resulted in cumulative defects in biofilm formation, similar to those seen in beta-lactamase-expressing transformants. Our results imply that horizontal acquisition of beta-lactamase resistance enzymes can have a phenotypic cost to bacteria by reducing their ability to form biofilms. Beta-lactamases likely affect PG remodelling, manifesting as perturbation of structures involved in bacterial adhesion that are required to initiate biofilm formation.

Use of in-biofilm expression technology to identify genes involved in Pseudomonas aeruginosa biofilm development.

Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness.

Pseudomonas aeruginosa cystic fibrosis clinical isolates produce exotoxin A with altered ADP-ribosyltransferase activity and cytotoxicity.

The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in the lung infections of cystic fibrosis (CF) patients is not well understood. Transcript-accumulation studies of bacterial populations in sputum reveal high levels of transcription of toxA, which encodes ETA, in some patients with CF. However, in general, tissue damage in the lungs of patients with CF does not seem to be consistent with a high level of expression of active ETA. To address this discrepancy the authors analysed the production and activity of ETA produced by a number of P. aeruginosa CF isolates. One CF isolate, strain 4384, transcribed toxA at levels similar to the hypertoxigenic strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADPRT) activity. Complementation in trans of strain 4384 with the wild-type toxA and a mixed toxin experiment suggested the absence of inhibitory accessory factors within this strain. The toxA gene from strain 4384 was cloned and sequenced, revealing only three mutations in the gene, all within the enzymic domain. The first mutation changed Ser-410 to Asn. The second mutation was located within an alpha-helix, altering Ala-476 to Glu. The third mutation, Ser-515 to Gly, was found at the protein surface. To date, Ser-410, Ala-476 and Ser-515 have not been reported to play a role in the ADPRT activity of ETA. However, it may be the combination of these mutations that reduces the enzymic activity of ETA produced by strain 4384. Expression of 4384 toxA and wild-type toxA in an isogenic strain revealed that 4384 ETA had 10-fold less ADPRT activity than wild-type ETA. ETA purified from strain 4384 also demonstrated 10-fold less ADPRT activity as compared to wild-type ETA. Cytotoxicity assays of purified ETA from strain 4384 indicated that the cytotoxicity of 4384 ETA is not reduced; it may be slightly more toxic than wild-type ETA. Analysis of five other CF isolates revealed a similar reduction in ADPRT activity to that seen in strain 4384. Sequence analysis of the enzymic domain of toxA from the five CF strains identified a number of mutations that could account for the reduction in ADPRT activity. These results suggest that some CF isolates produce an ETA with reduced enzymic activity and this may partially explain the pathogenesis of chronic lung infections of CF due to P. aeruginosa.