is one of the most common nosocomial pathogens worldwide, known for its virulence, drug resistance, and elaborate sensor-response network. The primary challenge encountered by pathogens during the ...initial stages of infection is the immune clearance arising from the host. The resident macrophages of barrier organs serve as the frontline defense against these pathogens. Central to our understanding is the mechanism by which bacteria modify their behavior to circumvent macrophage-mediated clearance, ensuring their persistence and colonization. To successfully evade macrophage-mediated phagocytosis, bacteria must possess an adaptive response mechanism. Two-component systems provide bacteria the agility to navigate diverse environmental challenges, translating external stimuli into cellular adaptive responses. Here, we report that the well-documented histidine kinase, LadS, coupled to a cognate two-component response regulator, PA0034, governs the expression of a vital adhesin called chaperone-usher pathway pilus cupA. The LadS/PA0034 system is susceptible to interference from the reactive oxygen species likely to be produced by macrophages and further lead to a poor adhesive phenotype with scantily cupA pilus, impairing the phagocytosis efficiency of macrophages during acute infection. This dynamic underscores the intriguing interplay: as macrophages deploy reactive oxygen species to combat bacterial invasion, the bacteria recalibrate their exterior to elude these defenses.
The notoriety of
is underscored by its virulence, drug resistance, and elaborate sensor-response network. Yet, the mechanisms by which
maneuvers to escape phagocytosis during acute infections remain elusive. This study pinpoints a two-component response regulator, PA0034, coupled with the histidine kinase LadS, and responds to macrophage-derived reactive oxygen species. The macrophage-derived reactive oxygen species can impair the LadS/PA0034 system, resulting in reduced expression of cupA pilus in the exterior of
. Since the cupA pilus is an important adhesin of
, its deficiency reduces bacterial adhesion and changes their behavior to adopt a planktonic lifestyle, subsequently inhibiting the phagocytosis of macrophages by interfering with bacterial adhesion. Briefly, reactive oxygen species may act as environmental cues for the LadS/PA0034 system. Upon recognition,
may transition to a poorly adhesive state, efficiently avoiding engulfment by macrophages.
Carbonic anhydrases (CAs) are involved in CO
uptake and conversion, a fundamental process in photosynthetic organisms. Nevertheless, the mechanism underlying the regulation of CO
uptake and ...intracellular conversion in cyanobacteria is largely unknown. We report the characterization of a previously unrecognized thylakoid-located CA Slr0051 (EcaB) from the cyanobacterium Synechocystis sp. PCC 6803, which possesses CA activity to regulate CO
uptake. Inactivation of ecaB stimulated CO
hydration in the thylakoids, suppressed by the classical CA inhibitor acetazolamide. Absence of ecaB increased the reduced state of the photosynthetic electron transport system, lowered the rate of photosynthetic O
evolution at high light (HL) and pH, and decreased the cellular affinity for extracellular inorganic carbon. Furthermore, EcaB was upregulated in cells grown at limiting CO
concentration or HL in tandem with CupA. EcaB is mainly located in the thylakoid membranes where it interacts with CupA and CupB involved in CO
uptake by converting it to bicarbonate. We propose that modulation of the EcaB level and activity in response to CO
changes, illumination or pH reversibly regulates its conversion to HCO
by the two CO
-uptake systems (CupA, CupB), dissipating the excess HCO
and alleviating photoinhibition, and thereby optimizes photosynthesis, especially under HL and alkaline conditions.
• Carbonic anhydrases (CAs) are involved in CO₂ uptake and conversion, a fundamental process in photosynthetic organisms. Nevertheless, the mechanism underlying the regulation of CO₂ uptake and ...intracellular conversion in cyanobacteria is largely unknown.
• We report the characterization of a previously unrecognized thylakoid-located CA Slr0051 (EcaB) from the cyanobacterium Synechocystis sp. PCC 6803, which possesses CA activity to regulate CO₂ uptake.
• Inactivation of ecaB stimulated CO₂ hydration in the thylakoids, suppressed by the classical CA inhibitor acetazolamide. Absence of ecaB increased the reduced state of the photosynthetic electron transport system, lowered the rate of photosynthetic O₂ evolution at high light (HL) and pH, and decreased the cellular affinity for extracellular inorganic carbon. Furthermore, EcaB was upregulated in cells grown at limiting CO₂ concentration or HL in tandem with CupA. EcaB is mainly located in the thylakoid membranes where it interacts with CupA and CupB involved in CO₂ uptake by converting it to bicarbonate.
• We propose that modulation of the EcaB level and activity in response to CO₂ changes, illumination or pH reversibly regulates its conversion to HCO₃ by the two CO₂-uptake systems (CupA, CupB), dissipating the excess HCO₃⁻ and alleviating photoinhibition, and thereby optimizes photosynthesis, especially under HL and alkaline conditions.
Pseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular ...aggregates. Cellular aggregates may occur attached to a surface (biofilm), at the air-liquid interface (pellicle), or as suspended aggregates. Compared to surface attached communities, knowledge about the regulatory processes involved in the formation of suspended cell aggregates is still limited. We have recently described the SiaA/D signal transduction module that regulates macroscopic cell aggregation during growth with, or in the presence of the surfactant SDS. Targets for SiaA/D mediated regulation include the Psl polysaccharide, the CdrAB two-partner secretion system and the CupA fimbriae. While the global regulators c-di-GMP and RsmA are known to inversely coordinate cell aggregation and regulate the expression of several adhesins, their potential impact on the expression of the cupA operon remains unknown. Here, we investigated the function of SiaA (a putative ser/thr phosphatase) and SiaD (a di-guanylate cyclase) in cupA1 expression using transcriptional reporter fusions and qRT-PCR. These studies revealed a novel interaction between the RsmA posttranscriptional regulatory system and SiaA/D mediated macroscopic aggregation. The RsmA/rsmY/Z system was found to affect macroscopic aggregate formation in the presence of surfactant by impacting the stability of the cupA1 mRNA transcript and we reveal that RsmA directly binds to the cupA1 leader sequence in vitro. We further identified that transcription of the RsmA antagonist rsmZ is controlled in a SiaA/D dependent manner during growth with SDS. Finally, we found that the siaD transcript is also under regulatory control of RsmA and that overproduction of RsmA or the deletion of siaD results in decreased cellular cyclic di-guanosine monophosphate (c-di-GMP) levels quantified by a transcriptional reporter, demonstrating that SiaA/D connects c-di-GMP and RsmA/rsmY/Z signaling to reciprocally regulate cell aggregation in response to environmental conditions.
Pseudomonas aeruginosa, an opportunistic pathogen, is a potent biofilm forming organism. Independent of the type IV pili, the cupA cluster in Pseudomonas aeruginosa facilitates biofilm formation ...which provides protection against antibacterial treatment. The pel and psl genes synthesizing exopolysaccharides also initiate biofilm formation. Investigations revealed antibiotic exposure to be associated with modulated expression of many resistance genes in different species of bacteria depending upon the concentrations in which these are used. In recent days, the use of carbapenems, is growing at an alarming rate to treat infections and may also have a regulatory effect in the expression of cupA, pslA and pelA genes associated with biofilm formation. The present study therefore was taken up to investigate the possible role of carbapenem on these gene's expression at sub lethal dose.
Six carbapenem non susceptible isolates of Pseudomonas aeruginosa of clinical origin were selected for the study. The isolates were cultured upto log phase in fresh Luria Bertani broth with and without 2 μg/ml imipenem and meropenem respectively and the transcriptional response of the cupA1, cupA2, cupA3, cupA4,cupA5, pelA and pslA genes in the isolates were studied by quantitative real time PCR using the ΔΔct method.
The analysis of transcriptional response of cupA1, cupA2, cupA3, cupA4,cupA5,pslA and pelA genes in the isolates revealed that the response was enhanced multiple folds under sub inhibitory concentration (2 μg/ml) imipenem and meropenem stress as compared to the response of the genes under normal growth condition i.e. without any antibiotic pressure.
The present study could underscore the role of imipenem and meropenem at sub lethal dose and could infer that both imipenem and meropenem at sub lethal dose has a regulatory effect on the transcriptional response of cupA, pelA and pslA genes which are involved in biofilm formation.
•Sub-inhibitory concentration of carbapenem may act as signals to enhance response of genes essential for biofilm formation•Transcriptional response of cupA genes enhanced at sub-inhibitory concentration (2 μg/ml) carbapenem•Transcriptional response of pslA and pelA genes enhanced at sub-inhibitory concentration (2 μg/ml) carbapenem
is identified as a versatile opportunistic microorganism with metabolic diversity contributing to a wide range of health burdens, especially in immunocompromised patients. This bacterium is the ...cause of 10 to 20% of nosocomial infections. In this study, we evaluated the phenotypic characterizations of biofilm formation in
clinical isolates using micro-titer plate assay. Indeed, we estimated the prevalence of QS (
,
,
,
,
,
) and virulence genes (
and
) by PCR. The results showed that among 69% of the isolates forming biofilm, 9% were strong biofilm producers, whereas 13% and 47% of isolates produced moderate and low amounts of biofilm, respectively. All isolates possessed
and seven QS genes (
,
,
,
,
,
,
), while 92% of the isolates possessed the
gene. Identification of these genes and their association with biofilm formation can be advantageous in adopting therapeutic methods.
The Steroids for Corneal Ulcers Trial (SCUT) was a multicenter, international study of bacterial keratitis in which 101 Pseudomonas aeruginosa infections were treated. Twenty-two of 101 P. aeruginosa ...isolates collected had a colony morphology characteristic of a loss-of-function mutation in lasR, the gene encoding a quorum-sensing master regulator. Ulcers caused by these 22 strains were associated with larger areas of corneal opacification, worse vision, and a lower rate of vision recovery in response to treatment than ulcers caused by the other isolates. The lasR sequences from these isolates each contained one of three nonsynonymous substitutions, and these strains were deficient in production of LasR-regulated protease and rhamnolipids. Replacement of lasR with either of the two most common lasR alleles from the SCUT isolates was sufficient to decrease protease and rhamnolipid production in PA14. Loss of LasR function is associated with increased production of CupA fimbriae, and the LasR-defective isolates exhibited higher production of CupA fimbriae than LasR-intact isolates. Strains with the same lasR mutation were of the same multilocus sequence type, suggesting that LasR-deficient, environmental P. aeruginosa strains were endemic to the area, and infections caused by these strains were associated with worse patient outcomes in the SCUT study. (This study has been registered at ClinicalTrials.gov under registration no. NCT00324168.) IMPORTANCE The LasR transcription factor is an important regulator of quorum sensing in P. aeruginosa and positively controls multiple virulence-associated pathways. The emergence of strains with lasR loss-of-function alleles in chronic disease is well described and is thought to represent a specific adaptation to the host environment. However, the prevalence and virulence of these strains in acute infections remain unclear. This report describes observations revealing that lasR mutants were common among isolates from a large, multicenter clinical study of keratitis and were associated with worse clinical outcomes than LasR-intact strains despite reduced production of LasR-regulated factors. Additionally, these lasR mutants were closely related strains or clones, as determined by molecular analysis. Because bacterial keratitis is community acquired, these data indicate infection by endemic, LasR-deficient strains in the environment. These results suggest that the conventional paradigm regarding the role for LasR-mediated regulation of virulence is more complex than previously appreciated.
ABSTRACT The Steroids for Corneal Ulcers Trial (SCUT) was a multicenter, international study of bacterial keratitis in which 101 Pseudomonas aeruginosa infections were treated. Twenty-two of 101 P. ...aeruginosa isolates collected had a colony morphology characteristic of a loss-of-function mutation in lasR, the gene encoding a quorum-sensing master regulator. Ulcers caused by these 22 strains were associated with larger areas of corneal opacification, worse vision, and a lower rate of vision recovery in response to treatment than ulcers caused by the other isolates. The lasR sequences from these isolates each contained one of three nonsynonymous substitutions, and these strains were deficient in production of LasR-regulated protease and rhamnolipids. Replacement of lasR with either of the two most common lasR alleles from the SCUT isolates was sufficient to decrease protease and rhamnolipid production in PA14. Loss of LasR function is associated with increased production of CupA fimbriae, and the LasR-defective isolates exhibited higher production of CupA fimbriae than LasR-intact isolates. Strains with the same lasR mutation were of the same multilocus sequence type, suggesting that LasR-deficient, environmental P. aeruginosa strains were endemic to the area, and infections caused by these strains were associated with worse patient outcomes in the SCUT study. (This study has been registered at ClinicalTrials.gov under registration no. NCT00324168 .) IMPORTANCE The LasR transcription factor is an important regulator of quorum sensing in P. aeruginosa and positively controls multiple virulence-associated pathways. The emergence of strains with lasR loss-of-function alleles in chronic disease is well described and is thought to represent a specific adaptation to the host environment. However, the prevalence and virulence of these strains in acute infections remain unclear. This report describes observations revealing that lasR mutants were common among isolates from a large, multicenter clinical study of keratitis and were associated with worse clinical outcomes than LasR-intact strains despite reduced production of LasR-regulated factors. Additionally, these lasR mutants were closely related strains or clones, as determined by molecular analysis. Because bacterial keratitis is community acquired, these data indicate infection by endemic, LasR-deficient strains in the environment. These results suggest that the conventional paradigm regarding the role for LasR-mediated regulation of virulence is more complex than previously appreciated.
Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through ...series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1-A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.
The larger protein complexes of the cyanobacterial photosynthetic membrane of
Thermosynechoccus elongatus and
Synechocystis 6803 were studied by single particle electron microscopy after detergent ...solubilization, without any purification steps. Besides the “standard” L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from
Synechocystis Δ
cupA and Δ
cupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO
2 or 3% CO
2 indicates that the number of NDH-1MS particles is 30-fold higher under low-CO
2.
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