The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa

Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI).

A Novel Use of Allopurinol as A Quorum-Sensing Inhibitor in Pseudomonas aeruginosa

Pseudomonas aeruginosa can cause a variety of healthcare-associated infections by its arsenal of virulence factors. Virulence factor production is largely controlled by the cell-to-cell communication system termed quorum sensing (QS). Targeting QS may be a good approach to inhibit the production of virulence factors and attenuate pathogenicity without exerting selective stress on bacterial growth. This will greatly reduce the emergence of resistant mutants. In this work, we investigated the anti-virulence and anti-QS activities of the FDA-approved drug allopurinol against the P. aeruginosa PAO1 strain. Allopurinol at 200 µg/mL (1/10 MIC) significantly decreased the production of the QS-controlled Chromobacterium violaceum CV026 violet pigment violacein and other P. aeruginosa QS-controlled virulence factors phenotypically. Furthermore, allopurinol reduced the infiltration of P. aeruginosa and leucocytes and diminished the congestion in the liver and kidney tissues of infected mice. In silico study showed that allopurinol could compete with the autoinducers on binding to the receptors LasR and RhlR by hydrogen bonding. On the molecular level, qRT-PCR proved that allopurinol showed a significant downregulating effect on all tested QS-encoding genes that regulate virulence factor production. In summary, allopurinol is a promising QS inhibitor that may be useful in the future treatment of P. aeruginosa infection.

Xylitol Inhibits Growth and Blocks Virulence in Serratia marcescens

Serratia marcescens is an opportunistic nosocomial pathogen and causes wound and burn infections. It shows high resistance to antibiotics and its pathogenicity is mediated by an arsenal of virulence factors. Another therapeutic option to such infections is targeting quorum sensing (QS), which controls the expression of different S. marcescens virulence factors. Prevention of QS can deprive S. marcescens from its bacterial virulence without applying stress on the bacterial growth and facilitates the eradication of the bacteria by immunity. The objective of the current study is to explore the antimicrobial and antivirulence activities of xylitol against S. marcescens. Xylitol could inhibit the growth of S. marcescens. Sub-inhibitory concentrations of xylitol could inhibit biofilm formation, reduce prodigiosin production, and completely block protease activity. Moreover, xylitol decreased swimming motility, swarming motility and increased the sensitivity to hydrogen peroxide. The expression of rsmA, pigP, flhC, flhD fimA, fimC, shlA bsmB, and rssB genes that regulate virulence factor production was significantly downregulated by xylitol. In silico study showed that xylitol could bind with the SmaR receptor by hydrophobic interaction and hydrogen bonding, and interfere with the binding of the natural ligand with SmaR receptor. An in vivo mice survival test confirmed the ability of xylitol to protect mice against the virulence of S. marcescens. In conclusion, xylitol is a growth and virulence inhibitor in S. marcescens and can be employed for the treatment of S. marcescens wound and burn infections.

ToxR mediates the antivirulence activity of phenyl-arginine-β-naphthylamide to attenuate Vibrio cholerae virulence.

Multidrug efflux systems belonging to the resistance-nodulation-cell division (RND) family are ubiquitous in Gram negative bacteria and critical for antimicrobial resistance. This realization has led to efforts to develop efflux pump inhibitors (EPI) for use as adjuvants for antibiotic treatment of resistant organisms. However, the functions of RND transporters extend beyond antimicrobial resistance to include physiological functions that are critical for pathogenesis, suggesting that EPIs could also be used as antivirulence therapeutics. This was documented in the enteric pathogen Vibrio cholerae where EPIs were shown to attenuate the production of the critical virulence factors cholera toxin (CT) and the toxin coregulated pilus (TCP). In this study we investigated the antivirulence mechanism of action of the EPI phenyl-arginine-β-naphthylamide (PAβN) on V. cholerae. Using bioassays, we documented that PAβN inhibited virulence factor production in three epidemic V. cholerae isolates. Transcriptional reporter studies and mutant analysis indicated that PAβN initiated a ToxR-dependent regulatory circuit to activate leuO expression and that LeuO repressed the expression of the critical virulence activator aphA to attenuate CT and TCP production. The antivirulence activity of PAβN was found to be dependent on the ToxR periplasmic sensing domain suggesting that a feedback mechanism was involved in its activity. Collectively the data indicated that PAβN inhibited V. cholerae virulence factor production by activating a ToxR-dependent metabolic feedback mechanism to repress the expression of the ToxR virulence regulon. This suggests that efflux pump inhibitors could be used as antivirulence therapeutics for the treatment of cholera and perhaps other gram negative pathogens.

Too much of a good thing: Overproduction of virulence factors impairs cryptococcal pathogenicity

The regulation of virulence factor production and deployment is crucial for the establishment of microbial infection and subsequent pathogenesis. If these processes are not properly coordinated, the infecting pathogen is less likely to both survive the immune response and cause damage to the host. One key virulence factor of the opportunistic fungal pathogen Cryptococcus neoformans, which kills almost 200,000 people each year worldwide, is a polysaccharide capsule that surrounds the cell wall; this structure helps the fungal cells resist engulfment and elimination by host phagocytes. Another important virulence trait is the development of a giant (Titan) cell morphotype that increases fungal resistance to phagocytosis, oxidative stress, and antifungal treatment. We recently identified the transcription factor Pdr802 as essential for C. neoformans adaptation to and survival under host conditions both in vitro and in vivo (Reuwsaat et al., mBio, doi: 10.1128/mBio.03457-20). Cryptococci lacking Pdr802 display enlarged capsules and enhanced Titan cell production, along with dramatically reduced virulence in a mouse model of infection. These results demonstrate that more is not necessarily better when it comes to virulence factors. Instead, precise regulation of these traits, to avoid both under- and overexpression, is critical for the success of this pathogen as it faces the challenges imposed by the host environment.

Staphylococcus aureus Floating Biofilm Formation and Phenotype in Synovial Fluid Depends on Albumin, Fibrinogen, and Hyaluronic Acid

Biofilms are typically studied in bacterial media that allow the study of important properties such as bacterial growth. However, the results obtained in such media cannot take into account the bacterial localization/clustering caused by bacteria–protein interactions in vivo and the accompanying alterations in phenotype, virulence factor production, and ultimately antibiotic tolerance. We and others have reported that methicillin-resistant or methicillin-susceptible Staphylococcus aureus (MRSA or MSSA, respectively) and other pathogens assemble a proteinaceous matrix in synovial fluid. This proteinaceous bacterial aggregate is coated by a polysaccharide matrix as is characteristic of biofilms. In this study, we identify proteins important for this aggregation and determine the concentration ranges of these proteins that can reproduce bacterial aggregation. We then test this protein combination for its ability to cause marked aggregation, antibacterial tolerance, preservation of morphology, and expression of the phenol-soluble modulin (PSM) virulence factors. In the process, we create a viscous fluid that models bacterial behavior in synovial fluid. We suggest that our findings and, by extension, use of this fluid can help to better model bacterial behavior of new antimicrobial therapies, as well as serve as a starting point to study host protein–bacteria interactions characteristic of physiological fluids.

Pseudmonas cannabina pv. alisalensis TrpA Is Required for Virulence in Multiple Host Plants

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial leaf spot and blight of Brassicaceae and Poaceae. We previously identified several potential Pcal virulence factors with transposon mutagenesis. Among these a trpA mutant disrupted the tryptophan synthase alpha chain, and had an effect on disease symptom development and bacterial multiplication. To assess the importance of TrpA in Pcal virulence, we characterized the trpA mutant based on inoculation test and Pcal gene expression profiles. The trpA mutant showed reduced virulence when dip- and syringe-inoculated on cabbage and oat. Moreover, epiphytic bacterial populations of the trpA mutant were also reduced compared to the wild-type (WT). These results suggest that TrpA contributes to bacterial multiplication on the leaf surface and in the apoplast, and disease development. Additionally, several Brassicaceae (including Japanese radish, broccoli, and Chinese cabbage) also exhibited reduced symptom development when inoculated with the trpA mutant. Moreover, trpA disruption led to downregulation of bacterial virulence genes, including type three effectors (T3Es) and the phytotoxin coronatine (COR), and to upregulation of tryptophan biosynthesis genes. These results indicate that a trade-off between virulence factor production and Pcal multiplication with tryptophan might be regulated in the infection processes.

A Mutation Upstream of the rplN-rpsD Ribosomal Operon Downregulates Bordetella pertussis Virulence Factor Production without Compromising Bacterial Survival within Human Macrophages

ABSTRACT The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5′-untranslated region (5′-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis. These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways. IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis. Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.