Microbes produce a biofilm matrix consisting of proteins, extracellular DNA, and polysaccharides that is integral in the formation of bacterial communities. Historical studies of polysaccharides ...revealed that their overproduction often alters the colony morphology and can be diagnostic in identifying certain species. The polysaccharide component of the matrix can provide many diverse benefits to the cells in the biofilm, including adhesion, protection, and structure. Aggregative polysaccharides act as molecular glue, allowing the bacterial cells to adhere to each other as well as surfaces. Adhesion facilitates the colonization of both biotic and abiotic surfaces by allowing the bacteria to resist physical stresses imposed by fluid movement that could separate the cells from a nutrient source. Polysaccharides can also provide protection from a wide range of stresses, such as desiccation, immune effectors, and predators such as phagocytic cells and amoebae. Finally, polysaccharides can provide structure to biofilms, allowing stratification of the bacterial community and establishing gradients of nutrients and waste products. This can be advantageous for the bacteria by establishing a heterogeneous population that is prepared to endure stresses created by the rapidly changing environments that many bacteria encounter. The diverse range of polysaccharide structures, properties, and roles highlight the importance of this matrix constituent to the successful adaptation of bacteria to nearly every niche. Here, we present an overview of the current knowledge regarding the diversity and benefits that polysaccharide production provides to bacterial communities within biofilms.
The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on ...gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.
Bacteria live in social communities, where the ability to sense and respond to interspecies and environmental signals is critical for survival. We previously showed the pathogen Pseudomonas ...aeruginosa detects secreted peptides from bacterial competitors and navigates through interspecies signal gradients using pilus-based motility. Yet, it was unknown whether P. aeruginosa utilizes a designated chemosensory system for this behavior. Here, we performed a systematic genetic analysis of a putative pilus chemosensory system, followed by high-speed live-imaging and single-cell tracking, to reveal behaviors of mutants that retain motility but are blind to interspecies signals. The enzymes predicted to methylate (PilK) and demethylate (ChpB) the putative pilus chemoreceptor, PilJ, are necessary for cells to control the direction of migration. While these findings implicate PilJ as a bona fide chemoreceptor, such function had yet to be experimentally defined, as full-length PilJ is essential for motility. Thus, we constructed systematic genetic modifications of PilJ and found that without the predicted ligand binding domains or predicted methylation sites, cells lose the ability to detect competitor gradients, despite retaining pilus-mediated motility. Chemotaxis trajectory analysis revealed that increased probability and size of P. aeruginosa pilus-mediated steps towards S. aureus peptides, versus steps away, determines motility bias in wild type cells. However, PilJ mutants blind to interspecies signals take less frequent steps towards S. aureus or steps of equal size towards and away. Collectively, this work uncovers the chemosensory nature of PilJ, provides insight into how cell movements are biased during pilus-based chemotaxis, and identifies chemotactic interactions necessary for bacterial survival in polymicrobial communities, revealing putative pathways where therapeutic intervention might disrupt bacterial communication.
Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic ...fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.
Microbes often live in multispecies communities where interactions among community members impact both the individual constituents and the surrounding environment. Here, we developed a system to ...visualize interspecies behaviors at initial encounters. By imaging two prevalent pathogens known to be coisolated from chronic illnesses,
and
, we observed
can modify surface motility in response to secreted factors from
. Upon sensing
transitioned from collective to single-cell motility with an associated increase in speed and directedness - a behavior we refer to as 'exploratory motility'. Explorer cells moved preferentially towards
and invaded
colonies through the action of the type IV pili. These studies reveal previously undescribed motility behaviors and lend insight into how
senses and responds to other species. Identifying strategies to harness these interactions may open avenues for new antimicrobial strategies.
Cystic fibrosis (CF) patients chronically infected with both
and
have worse health outcomes than patients who are monoinfected with either
or
We showed previously that mucoid strains of
can coexist ...with
due to the transcriptional downregulation of several toxic exoproducts typically produced by
, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline
-oxide). Here, we demonstrate that exogenous alginate protects
from
in both planktonic and biofilm coculture models under a variety of nutritional conditions.
protection in the presence of exogenous alginate is due to the transcriptional downregulation of
, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (
quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid
with nonmucoid
strains can mitigate the killing of
by the nonmucoid strain of
, indicating that the mechanism that we describe here may function
in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill
at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the
-protective effects of mucoidy in a strain-specific manner.
CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are
and
, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote
-
coexistence, despite evidence that
kills
when these organisms are cocultured
Thus, a better understanding of
-
interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.
:
and
often infect the airways in cystic fibrosis (CF). Because registry studies show higher prevalence of
versus
in older patients with CF, a common assumption is that
replaces
over time.
,
can ...outgrow and kill
. However, it is unknown how rapidly
replaces
in patients with CF.
: We studied a longitudinal cohort of children and adults with CF who had quantitative sputum cultures. We determined the abundance of
and
in cfu/ml. We determined the duration and persistence of infections and measured longitudinal changes in culture positivity and abundance for each organism.
: Between 2004 and 2017, 134 patients had ≥10 quantitative cultures, with median observation time of 10.15 years. One hundred twenty-four patients had at least one positive culture for
, and 123 had at least one positive culture for
. Both species had median abundance of >10
cfu/ml. Culture abundance was stable over time for both organisms. There was an increase in the prevalence of
/
coinfection but no decrease in
prevalence within individuals over time.
:
and
are abundant in CF sputum cultures. Contrary to common assumption, we found no pattern of replacement of
by
. Many patients with CF have durable long-term coinfection with these organisms. New strategies are needed to prevent and treat these infections.
causes chronic pulmonary infections in patients with cystic fibrosis (CF).
mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF ...patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both
and
Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H
O
). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H
O
stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on
, encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of
mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung.
mucoid conversion within lungs of cystic fibrosis (CF) patients is a hallmark of chronic infection and predictive of poor prognosis. The selective benefit of mixed populations of mucoid and nonmucoid variants, often isolated from chronically infected CF patients, has not been explored. Here, we show that mixed-variant communities of
demonstrate advantages in evasion of innate antimicrobials via production of shared goods: alginate and catalase. These data argue for therapeutically targeting multiple constituents (both mucoid and nonmucoid variants) within diversified
communities
, as these variants can differentially shield one another from components of the host response.
Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, ...these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.
While complex intra- and interspecies microbial community dynamics are apparent during chronic infections and likely alter patient health outcomes, our understanding of these interactions is ...currently limited. For example,
and
are often found to coinfect the lungs of patients with cystic fibrosis (CF), yet these organisms compete under laboratory conditions. Recent observations that coinfection correlates with decreased health outcomes necessitate we develop a greater understanding of these interbacterial interactions. In this study, we tested the hypothesis that
and/or
adopts phenotypes that allow coexistence during infection. We compared competitive interactions of
and
isolates from mono- or coinfected CF patients employing
coculture models.
isolates from monoinfected patients were more competitive toward
than
isolates from coinfected patients. We also observed that the least competitive
isolates possessed a mucoid phenotype. Mucoidy occurs upon constitutive activation of the sigma factor AlgT/U, which regulates synthesis of the polysaccharide alginate and dozens of other secreted factors, including some previously described to kill
Here, we show that production of alginate in mucoid strains is sufficient to inhibit anti-
activity independent of activation of the AlgT regulon. Alginate reduces production of siderophores, 2-heptyl-4-hydroxyquinolone-
-oxide (HQNO), and rhamnolipids-each required for efficient killing of
These studies demonstrate alginate overproduction may be an important factor driving
coinfection with
Numerous deep-sequencing studies have revealed the microbial communities present during respiratory infections in cystic fibrosis (CF) patients are diverse, complex, and dynamic. We now face the challenge of determining the influence of these community dynamics on patient health outcomes and identifying candidate targets to modulate these interactions. We make progress toward this goal by determining that the polysaccharide alginate produced by mucoid strains of
is sufficient to inhibit multiple secreted antimicrobial agents produced by this organism. Importantly, these secreted factors are required to outcompete
, when the microbes are grown in coculture; thus we propose a mechanism whereby mucoid
can coexist with
Finally, the approach used here can serve as a platform to investigate the interactions among other CF pathogens.
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