Hongwei D. Yu

PBAD-Based Shuttle Vectors for Functional Analysis of Toxic and Highly Regulated Genes in Pseudomonas and Burkholderia spp. and Other Bacteria

ABSTRACT We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the PBAD promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.

Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity

ABSTRACT Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.

Regulated proteolysis controls mucoid conversion in Pseudomonas aeruginosa

Overproduction of the exopolysaccharide alginate causes mucoid conversion in Pseudomonas aeruginosa and is a poor prognosticator in cystic fibrosis. The ECF σ factor AlgU and its cognate anti-σ factor MucA are two principal regulators of alginate production. Here, we report the identification of three positive regulators of alginate biosynthesis: PA4033 (designated mucE), PA3649 (designated mucP), and algW. MucE, a small protein (9.5 kDa), was identified as part of a global mariner transposon screen for new regulators of alginate production. A transposon located in its promoter caused the overexpression of MucE and mucoid conversion in P. aeruginosa strains PAO1 and PA14. Accumulation of MucE in the envelope resulted in increased AlgU activity and reduced MucA levels. Three critical amino acid residues at the C terminus of MucE (WVF) were required for mucoid conversion via two predicted proteases AlgW (DegS) and MucP (RseP/YaeL). Moreover, as in Escherichia coli, the PDZ domain of AlgW was required for signal transduction. These results suggest that AlgU is regulated similarly to E. coli σE except that the amino acid triad signals from MucE and other envelope proteins that activate AlgW are slightly different from those activating DegS.

Comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens

Segmented filamentous bacteria (SFB) are indigenous gut commensal bacteria. They are commonly detected in the gastrointestinal tracts of both vertebrates and invertebrates. Despite the significant role they have in the modulation of the development of host immune systems, little information exists regarding the presence of SFB in humans. The aim of this study was to investigate the distribution and diversity of SFB in humans and to determine their phylogenetic relationships with their hosts. Gut contents from 251 humans, 92 mice and 72 chickens were collected for bacterial genomic DNA extraction and subjected to SFB 16S rRNA-specific PCR detection. The results showed SFB colonization to be age-dependent in humans, with the majority of individuals colonized within the first 2 years of life, but this colonization disappeared by the age of 3 years. Results of 16S rRNA sequencing showed that multiple operational taxonomic units of SFB could exist in the same individuals. Cross-species comparison among human, mouse and chicken samples demonstrated that each host possessed an exclusive predominant SFB sequence. In summary, our results showed that SFB display host specificity, and SFB colonization, which occurs early in human life, declines in an age-dependent manner.

ClpXP proteases positively regulate alginate overexpression and mucoid conversion in Pseudomonas aeruginosa

Overproduction of the exopolysaccharide alginate and conversion to a mucoid phenotype in Pseudomonas aeruginosa are markers for the onset of chronic lung infection in cystic fibrosis (CF). Alginate production is regulated by the extracytoplasmic function (ECF) sigma factor AlgU/T and the cognate anti-sigma factor MucA. Many clinical mucoid isolates carry loss-of-function mutations in mucA. These mutations, including the most common mucA22 allele, cause C-terminal truncations in MucA, indicating that an inability to regulate AlgU activity by MucA is associated with conversion to the mucoid phenotype. Here we report that a mutation in a stable mucoid strain derived from the parental strain PAO1, designated PAO581, that does not contain the mucA22 allele, was due to a single-base deletion in mucA (DeltaT180), generating another type of C-terminal truncation. A global mariner transposon screen in PAO581 for non-mucoid isolates led to the identification of three regulators of alginate production, clpP (PA1801), clpX (PA1802), and a clpP paralogue (PA3326, designated clpP2). The PAO581 null mutants of clpP, clpX and clpP2 showed decreased AlgU transcriptional activity and an accumulation of haemagglutinin (HA)-tagged N-terminal MucA protein with an apparent molecular mass of 15 kDa. The clpP and clpX mutants of a CF mucoid isolate revert to the non-mucoid phenotype. The ClpXP and ClpP2 proteins appear to be part of a proteolytic network that degrades the cytoplasmic portion of truncated MucA proteins to release the sequestered AlgU, which drives alginate biosynthesis.

Pseudomonas aeruginosa in cystic fibrosis: role of mucC in the regulation of alginate production and stress sensitivity

Alginate production in Pseudomonas aeruginosa and the associated mucoid phenotype of isolates from cystic fibrosis patients are under the control of the algU mucABCD cluster. This group of genes encodes AlgU, the P. aeruginosa equivalent of the extreme heat shock sigma factor sigma E in Gram-negative bacteria, the AlgU-cognate anti-sigma factor MucA, the periplasmic protein MucB and a serine protease homologue, MucD. While mucA, mucB or mucD act as negative regulators of AlgU, the function of mucC is not known. In this study the role of mucC in P. aeruginosa physiology and alginate production has been addressed. Insertional inactivation of mucC in the wild-type P. aeruginosa strain PAO1 did not cause any overt effects on alginate synthesis. However, it affected growth of P. aeruginosa under conditions of combined elevated temperature and increased ionic strength or osmolarity. Inactivation of mucC in mucA or mucB mutant backgrounds resulted in a mucoid phenotype when the cells were grown under combined stress conditions of elevated temperature and osmolarity. Each of the stress factors tested separately did not cause comparable effects. The combined stress factors were not sufficient to cause phenotypically appreciable enhancement of alginate production in mucA or mucB mutants unless mucC was also inactivated. These findings support a negative regulatory role of mucC in alginate production by P. aeruginosa, indicate additive effects of muc genes in the regulation of mucoidy in this organism and suggest that multiple stress signals and recognition systems participate in the regulation of algU-dependent functions.

The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis

Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-sigma factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P(algU) and P(algD). Deletion of alternative sigma factor RpoN (sigma(54)) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

Analysis of the Pseudomonas aeruginosa Regulon Controlled by the Sensor Kinase KinB and Sigma Factor RpoN

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.

Pseudomonas aeruginosa MucD Regulates the alginate Pathway through Activation of MucA Degradation via MucP Proteolytic Activity

Alginate overproduction in Pseudomonas aeruginosa can be caused by the proteolysis of the anti-sigma factor MucA regulated by the AlgW protease. Here, we show that inactivation of MucD, an HtrA/DegP homolog and alginate regulator, can bypass AlgW, leading to an atypical proteolysis of MucA that is dependent on the MucP protease.

Persistent infections and immunity in cystic fibrosis.

Cystic fibrosis (CF) is the most common autosomal recessive lethal disease in the Caucasian population. Chronic respiratory infections with Pseudomonas aeruginosa, neutrophil-dominated airway inflammation and progressive lung damage are the major causes of morbidity and mortality in CF. Two persistent infection phenotypes expressed by this bacterium are biofilm and mucoidy. Biofilm, also called the microcolony mode of growth is the surface-associated adherent bacterial community, while mucoidy refers to a phenotype conducive to copious amounts of mucoid exopolysaccharide (MEP)/alginate that provides a matrix for mature biofilms conferring resistance to host defenses and antibiotics. Recent completion of the whole genomic sequence of the standard reference strain P. aeruginosa PAO1 has led to discoveries that many clinical isolates of this species possess unique genomic sequences (genomic islands) due to horizontal gene transfer. We propose this type of genetic exchange may play an important role in causing intrinsic genomic diversity of this organism. Therefore, the diversity, as revealed through profiles of restriction fragment length polymorphism (RFLP), may be linked to an array of novel and unexplored pathogenic mechanisms in P. aeruginosa. CF mouse models, while displaying many clinical similarities to human CF, have yet to demonstrate a chronic pulmonary disease phenotype. This review is intended to provide an overview of P. aeruginosa persistent infection phenotypes (biofilm and mucoidy) and an aerosol infection mouse model for CF. Genomic diversity of P. aeruginosa and its implications in the pathogenesis in CF will also be discussed.