INTERBACTERIAL COMPETITION IN THE BURKHOLDERIA CEPACIA COMPLEX
Andrew Irving Perault
A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor in Philosophy in the Department of
Microbiology and Immunology in the School of Medicine.
Chapel Hill 2020
Ó 2020
ABSTRACT
Andrew Irving Perault: Interbacterial Competition in the Burkholderia cepacia Complex (Under the direction of Peggy A. Cotter)
Bacteria often live in multicellular communities composed of multiple species. Bacteria interact with one another during intimate cell-cell contact within such environments, and these interactions influence the ecology and evolution of multicellular communities. Polymicrobial communities reside within the respiratory tracts of individuals with the genetic disorder cystic fibrosis (CF). Polymicrobial respiratory infections are characteristic of CF disease, and canonical pathogens can dominate the airway communities during infections. Intriguingly, pathogenic species of the Burkholderia cepacia complex (Bcc) can establish infections within polymicrobial CF respiratory tracts, even those dominated by the prevalent pathogen Pseudomonas
aeruginosa, though these infections do not occur in young individuals and are limited to teenage and adult CF patients. Here we describe two mechanisms of cell contact-dependent
interbacterial competition that provide Bcc pathogens ecological advantages: contact-dependent growth inhibition (CDI) and the type VI secretion system (T6SS).
variety of target bacteria. We describe the striking ability for this pathogen to use its T6SS to outcompete P. aeruginosa isolates from teenage and adult CF patients, but not P. aeruginosa isolates from young patients. Whole genome sequencing identified mutations in the susceptible P. aeruginosa isolates that abrogate activity by their own T6SSs, thus inhibiting their retaliation to attack from B. cenocepacia and promoting their elimination from co-cultures. These
ACKNOWLEDGEMENTS
I am fortunate to have wonderful support circles, both professionally and
non-professionally, and therefore a plethora of individuals deserve my gratitude. For the sake of brevity, I will try to keep this brief…
First, I would like to thank my mentor Peggy A. Cotter. Academia can be a welcoming and supportive community, and graduate student trainees can be blessed by having terrific mentors; however, no one is luckier than me and the fellow students to be trained by Peggy. Forget her passion for science, her excitement to discuss data, her love for helping trainees become great scientists, her ability to let loose; Peggy is just a great person. To get that and all the professional benefits with a mentor, what else could you ask for? I was lucky to have a similarly great mentor, Victor J. DiRita, before starting my graduate studies at UNC, and I thank him as well. I also would like to thank my thesis committee members. Although we only have formal meetings once a year, your mentorship and assistance in progressing my thesis research certainly extends beyond that. I also want to thank my unofficial mentors within the UNC
Microbiology & Immunology community and all the amazing individuals and support staff we have in our department.
great fun in scientific research, but I have also been lucky to have great fun outside of lab, thanks to absolutely wonderful friends I have made over the years. My friends in North Carolina, including my graduate school colleagues, non-scientist friends, and all those who were
TABLE OF CONTENTS
LIST OF FIGURES ... ix
LIST OF TABLES ... xi
LIST OF ABBREVIATIONS ... xii
CHAPTER 1 – Introduction ... 1
1.1 Microbial Communities ... 1
1.2 Microbial Cooperation within Communities ... 2
1.3 Microbial Competition within Communities ... 2
1.4 Contact-Dependent Growth Inhibition ... 3
1.5 Type VI Secretion Systems ... 7
1.6 Polymicrobial Communities within the Cystic Fibrosis Respiratory Tract ... 10
1.7 Pseudomonas aeruginosa ... 12
1.8 The Burkholderia cepacia Complex ... 15
1.9 Objectives of this Dissertation ... 18
1.10 References ... 21
CHAPTER 2 – Three Distinct Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition by the Cystic Fibrosis Pathogen Burkholderia dolosa ... 40
2.1 Summary ... 40
2.2 Importance ... 41
2.3 Introduction ... 41
2.4 Materials and Methods ... 43
2.5 Results ... 48
2.6 Discussion ... 56
2.7 References ... 73
3.1 Summary ... 78
3.2 Introduction ... 78
3.3 Results ... 80
3.4 Discussion ... 88
3.5 Method Details ... 94
3.6 References ... 114
CHAPTER 4 – Conclusion ... 122
4.1 Interbacterial Competition in the Context of Cystic Fibrosis Infections ... 122
4.2 My Contributions to the Burkholderia cepacia Complex Sociomicrobiology Field ... 123
4.3 CDI System-Mediated Competition and Cooperation by the Burkholderia cepacia Complex ... 124
4.4 T6SS-Mediated Burkholderia cepacia Complex Colonization and Infection of the Cystic Fibrosis Respiratory Tract ... 127
4.5 Future Investigations for the Burkholderia cepacia Complex Sociomicrobiology Field .. 132
4.6 References ... 135
LIST OF FIGURES
Figure 1.1. CDI Model ... 19
Figure 1.2. T6SS model ... 20
Figure 2.1. bcpAIOB loci in BdAU0158 ... 62
Figure 2.2. The Bcp-1 and Bcp-2 CDI systems provide BdAU0158 a competitive advantage during in vitro growth ... 63
Figure 2.3. The BdAU0158 bcp-3 locus encodes a functional CDI system that is not expressed under in vitro competition conditions ... 64
Figure 2.4. BdAU0158 BcpO proteins are not required for CDI-mediated competition or resistance to CDI ... 65
Figure 2.5. The Bcp-dependent community behaviors of autoaggregation and pigment production are not evident in BdAU0158 ... 66
Figure 2.6. E. coli autotoxicity due to inducible production of the BdAU0158 Bcp-2 and Bcp-3 CT toxins, but not the Bcp-1 CT toxin ... 67
Figure 2.7. The BdAU0158 Bcp-1 allele contains the same CT toxin- immunity pair as the BtE264 Bcp allele ... 68
Figure 2.8. Orthologs of the BdAU0158 BcpA-CT and BcpI across several Burkholderia strains ... 69
Figure 2.9. Production of BdAU0158 BcpA-1-CT and BcpI-1 by E. coli ... 70
Figure 3.1. The BcAU1054 T6SS mediates interbacterial competition ... 100
Figure 3.2. The BcAU1054 T6SS core cluster is homologous to the BcJ2315 T6SS core cluster, though contains insertions ... 101
Figure 3.3. BcAU1054 ∆hcp does not have a growth defect ... 102
Figure 3.4. Screening approach to identify probable BcAU1054 T6SS E-I pairs ... 103
Figure 3.5. At least five BcAU1054 T6SS effectors mediate interbacterial competition ... 104
Figure 3.6. Susceptibility of P. aeruginosa CF isolates to the BcAU1054 T6SS correlates with patient age ... 105
Figure 3.7. BcAU1054 T6SS effectors exhibit target strain-specific variability in toxicity ... 106
Figure 3.8. Restoration of H1-T6SS protein production can rescue host- adapted P. aeruginosa from T6SS-mediated elimination by BcAU1054 ... 107
LIST OF TABLES
Table 2.1. Bacterial strains used in this study ... 71 Table 2.2 Plasmids used in this study ... 72 Table 3.1. Bioinformatically-predicted BcAU1054 T6SS E-I pairs and
predicted effector enzymatic activities ... 110 Table 3.2. Competition sensitivity, Hcp1 production, and putative T6SS-
LIST OF ABBREVIATIONS
ATPase adenosine triphosphatase
BcAU1054 Burkholderia cenocepacia strain AU1054 Bcc Burkholderia cepacia complex
BdAU0158 Burkholderia dolosa strain AU0158 BmCGD2M Burkholderia multivorans strain CGD2M
bp base pair
BtE264 Burkholderia thailandensis strain E264
C.I. competitive index
CDI contact-dependent growth inhibition CDS contact-dependent signaling
CF cystic fibrosis
CFTR cystic fibrosis transmembrane conductance regulator CGD chronic granulomatous disease
Cm chloramphenicol
CT C terminus
DNA deoxyribonucleic acid E-I effector-immunity
GTPase guanosine triphosphatase
IM inner membrane
kDa kilodalton
Km kanamycin
LB lysogeny broth
mRNA messenger ribonucleic acid
NT N terminus
OD600 optical density, 600 nm
OM outer membrane
ORF open reading frame PBS phosphate-buffered saline PCR polymerase chain reaction RNA ribonucleic acid
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis sRNA small regulatory RNA
Tet tetracycline
TPS two-partner secretion tRNA transfer ribonucleic acid T3SS type III secretion system T5SSb type Vb secretion system T6S type VI secretion
T6SS type VI secretion system
CHAPTER 1 – INTRODUCTION
1.1 Microbial Communities
Bacteria are social organisms often residing within multicellular communities made up of one or many species (1, 2). These communities, typically referred to as biofilms, can be free-floating in aqueous environments or surface-attached, and are usually encased in an
extracellular matrix composed of secreted macromolecules including polysaccharides, DNA, proteins, and lipids (3, 4). Due to the matrix surrounding biofilms, bacterial cells aggregate and can be in intimate cell-cell contact, promoting direct interactions between cells. Bacteria can also interact from afar within biofilms via secreted molecules (3, 5). These interactions have dramatic effects on the ecology and evolution of biofilm residents (6, 7).
Microbial communities are instrumental to our natural world. From their impact on biogeochemical cycling (8, 9) to their essentiality for plant growth (10), microbial communities influence everyday life. Humans are colonized by microbial communities at several body sites, including the skin, oral cavity, intestinal tract, and the female reproductive tract, where these bacteria are required for proper health of their host (11-14). Alternatively, human-associated microbial communities can have deleterious effects on their hosts and promote disease.
1.2 Microbial Cooperation within Communities
Bacteria form beneficial interactions with kin cells and cells of different microbial strains and species within biofilms (20). Secreted molecules in the form of “public goods” improve nutrient acquisition by community members (21, 22), and bacteria digest complex
macromolecules that feed into central metabolism more efficiently within biofilms compared to cells in isolation (23, 24). Human gut Bacteroides spp. are adept at degrading complex
polysaccharides and releasing smaller molecules that serve as energy sources for other members of the intestinal microbiota (25). Additional secreted “public goods” enhance surface attachment and retention within the community (26, 27), as well as mediate communication and cooperative behaviors among cells (28). Cells unable to produce biofilm matrix components can be incorporated into a growing biofilm as long as matrix-producers are present (29). Due to the extracellular matrix and gradients of nutrients and oxygen, the physical nature of biofilms provides a protective shield against cellular stressors including antibiotics (19), and thus biofilm formation can be a cooperative behavior benefiting all community members.
1.3 Microbial Competition within Communities
Converse to the potential collective benefits of cooperative behaviors within biofilms is the high degree of intercellular competition that occurs in these communities (30-32).
metabolites produced by microorganisms that can diffuse throughout the environment and kill competitor cells (36). Degradative enzymes also act as diffusible antimicrobial weapons and provide competitive advantages to several bacterial species, including Myxococcus xanthus (37) and Staphylococcus spp. (38). Certain bacteria bleb off portions of their cellular membranes to form extracellular vesicles, which can deliver toxic cargo to competitor bacteria (39).
Pseudomonas aeruginosa (40), Streptomyces spp. (41, 42), M. xanthus (43), and Lysobacter (44) have been shown to kill competitor bacteria by delivering toxins within extracellular vesicles.
Interbacterial competition dependent on intimate cell-cell contact can be mediated by complex secretory systems or by direct transfer of membrane components (32, 35). In addition to the battery of secreted antimicrobials produced by M. xanthus, this species delivers
polymorphic toxins to Myxococcus target cells during an outer membrane (OM) fusion process termed “outer membrane exchange” (45, 46). Specialized secretion systems, including the type IV, type Vb, type VI, and type VII secretion systems (T4SS, T5SSb, T6SS, and T7SS,
respectively) also act as potent antibacterial weapons. The T4SS, T5SSb, and T6SS are widespread throughout Gram-negative bacteria (32), though T4SS-mediated interbacterial competition has only been described in the species Xanthomonas citri (47). The T7SS is a newly-characterized mechanism of interbacterial competition found in Gram-positive bacteria (48, 49). Of the specialized secretion systems mediating cell contact-dependent interbacterial competition, the T5SSb (also referred to as contact-dependent growth inhibition, or CDI) and the T6SS are the most characterized.
1.4 Contact-Dependent Growth Inhibition
CDI systems are composed of proteins belonging to the two-partner secretion (TPS) pathway proteins (also called T5SSb proteins) (50). CDI was first discovered in rat fecal
cell-cell contact (51). Further investigations revealed genes predicted to encode CDI systems are widespread in Proteobacteria and that a key component of these systems are immunity
proteins, which protect against CDI toxins (52, 53). CDI systems fall into two broad classes: the E. coli-type systems and the Burkholderia-type systems. E. coli-type systems are found in a-, b-,
and g-proteobacteria, whereas Burkholderia-type systems are only found in members of the Burkholderia genus and other closely-related species (52, 53). The major discerning
characteristic between the two CDI classes is their operon layout. E. coli-type systems are encoded by cdiBAI loci (51), and Burkholderia-type systems are encoded by bcpAIOB loci (53). bcpO genes are present only in some Burkholderia-type CDI system-encoding loci, and the amino acid sequences of their encoded proteins are conserved across alleles (except for the signal sequences) (53, 54). No clear role exists for BcpO proteins, though B. thailandensis strain E264 (BtE264) and B. dolosa strain AU0158 (BdAU0158) mutants lacking the bcpO genes show a diminished ability to kill target cells via CDI (53, 54).
Despite their differences in genetic organization, E. coli-type and Burkholderia-type CDI systems function similarly. The CdiB/BcpB proteins are the transporter proteins of the TPS system, forming b barrels in the OM and allowing for translocation of the CdiA/BcpA passenger proteins (50). The CdiA/BcpA proteins are large exoproteins (typically >3000 amino acids) containing filamentous hemagglutinin repeats forming rigid b-helical rods on the surfaces of cells (Figure 1.1) (50). The C-terminal portions of CdiA/BcpA comprise the antibacterial toxins of CDI systems (52). Conserved amino acid motifs (VENN in E. coli-type systems, Nx(E/Q)LYN
from CDI-mediated killing by the same kind (i.e., cells that have the same CDI system-encoding allele) (52, 53, 55). The amino acid sequences of C-terminal toxins (CdiA-CTs and BcpA-CTs) and their cognate CdiI/BcpI proteins co-vary, resulting in polymorphic toxin-antitoxin protein pairs (52, 53). Of the CdiA-CTs and BcpA-CTs characterized to date, most have either been shown to be or are predicted to function as nucleases, degrading DNA or tRNA within target cells (54, 56-61).
Both E. coli-type and Burkholderia-type CDI systems can function modularly. The DNA sequence encoding the CT of one allele can be replaced with DNA sequence encoding another allele’s CT, and these chimeric CdiA/BcpA proteins still function in CDI and toxicity is only prevented by production of the CdiI/BcpI protein cognate to the chimeric CdiA-CT/BcpA-CT (52, 55). Given the modular capabilities and the polymorphic nature of CDI systems, homologous recombination may promote diversification of these systems by swapping in alternative toxin-immunity-encoding sequences (62). Certain Burkholderia-type CDI system-encoding genes are predicted to be located in mobile genetic islands (63, 64), including the BtE264 bcpAIOB genes, which are mobile via transposon-mediated insertion (65). Therefore, homologous recombination and transposition may be common mechanisms to diversify and disseminate CDI system-encoding genes throughout microbial communities.
Recent findings have updated the predicted model of CDI toxin delivery into target cells. Using EC93 as a model organism, the CdiA-CT was shown to remain in the periplasm of the cell, whereas the majority of the N-terminal conserved region, including the predicted receptor binding domain, was extracellular, forming a loop to thread the C terminus into the periplasm. It was proposed that upon CdiA binding to an OM receptor on the target cell, the C terminus would be secreted out of the periplasm and the CdiA-CT toxin would be delivered to the target cell (Figure 1.1) (66). While the OM receptor allowing for CDI toxin translocation into target cells has not been identified for Burkholderia-type CDI systems, the OM proteins BamA and
(67, 68). Additionally, inner membrane (IM) translocator proteins, often ABC transporter
membrane permeases, facilitate CdiA-CT delivery into the target cell cytoplasm where the toxin can elicit its effects (for nuclease toxins) (69). “Permissive factors” within target cells have also been identified that are required for toxicity of the delivered E. coli-type CDI toxins (57, 70, 71). Further investigations into the OM receptors for CdiA proteins revealed that the surface exposed loops 6 and 7 of BamA and the surface-exposed loops 4 and 5 of OmpC are the domains that interact with an inhibitor cell’s CdiA, and that the amino acid sequences of these loops covary with the sequences of the predicted receptor-binding domains on CdiA proteins (68, 72-74). These results suggested that a cell producing an E. coli-type CDI system can likely only deliver CdiA-CT toxins to phylogenetically related target cells, possibly cells so closely related that they encode the same cdiBAI allele, and thus would be immune to toxicity. Moreover, the IM
translocator proteins and “permissive factors” may add additional levels of target cell specificity for CdiA proteins and restrict potential CDI-mediated toxicity to closely related target cells, bringing into question the true ecological role of CDI systems in microbial communities.
intercellular communication was termed contact-dependent signaling (CDS) (76). BdAU0158, a human pathogenic member of the Burkholderia cepacia complex (Bcc), shares a BcpAIOB system almost identical to that of BtE264, and this BdAU0158 BcpA protein promoted similar gene expression changes in BtE264 within BdAU0158-BtE264 co-cultures (76); moreover, BdAU0158 and another Bcc pathogen Burkholderia multivorans produce functional CDI systems that kill target cells (54, 77). CDI system-mediated interbacterial competition and
communication/cooperation may be widespread in Burkholderia spp.
1.5 Type VI Secretion Systems
T6SSs mediate interbacterial competition by delivering proteinaceous effectors to target cells during secretion events. Effectors associate with either Hcp or VgrG proteins of the T6S apparatus (91, 92), and “evolved” VgrGs contain their own effector domains (79, 80, 92, 93). The T6SS was first described as an anti-eukaryotic weapon (79), and many bacterial pathogens deliver effectors into host cells using T6S (94); however, T6SSs are predicted to be produced by ~25% of all Gram-negative bacteria (95), many of which are not pathogens, and thus
interbacterial competition is believed to be the true ecological and evolutionary role of these systems (96). Antibacterial T6SS effectors have diverse molecular targets within prey cells, including membrane phospholipids, cell wall peptidoglycan, and nucleic acids (97). Recently characterized effectors also inhibit cell division by targeting FtsZ ring formation (98) and induce the stringent response by synthesizing (p)ppApp (99). Similar to CDI toxins, T6SS effectors have cognate antitoxins called immunity proteins, which are encoded by genes in immediate proximity to their cognate effector-encoding genes, composing effector-immunity (E-I)-encoding gene pairs. Immunity proteins protect against autotoxicity in T6SS-producing cells, as well as protect against kind-cell killing (97).
In contrast to CDI systems, target cell specificity is not known to restrict T6SS-mediated competition to closely related cells. Interspecies competition via T6S, including between
members of different proteobacterial classes, has been documented (78, 100, 101), though T6SS targeting of Gram-positive bacteria is not known to occur (102), perhaps due to the thick Gram-positive cell wall acting as a shield against these antibacterial weapons. Studies using the Pseudomonas aeruginosa reference strain PAO1, which produces three unique T6SSs, have
favorably target antagonistic competitor bacteria, at least for PAO1; however, it remains unknown whether other bacterial species exhibit similar specific T6SS activity.
Several studies have described T6SS-mediated interbacterial competition within higher host organisms. The plant pathogen Agrobacterium tumefaciens outcompetes bacterial targets via T6SS-activity during plant infection (104), and the plant commensal Pseudomonas putida uses its T6SS to outcompete multiple phytopathogens during plant colonization experiments (105). Several human pathogens have been shown to outcompete bacterial targets in a T6SS-dependent manner during murine model infection, including Salmonella enterica serovar Typhimurium (106), Shigella sonnei (107), and Vibrio cholerae (108), and T6SS-mediated competition improves the colonization of these pathogens. Human gut commensal Bacteroides spp. also use T6SSs for interbacterial competition and to gain a competitive advantage during intestinal colonization (109, 110). Moreover, metagenomic analyses of the human intestinal microbiota suggest T6S provides Bacteroides spp. and additional Bacteroidales spp. competitive advantages in the human gut (111, 112).
Although interbacterial competition is proposed to be the primary role of T6SSs,
1.6 Polymicrobial Communities within the Cystic Fibrosis Respiratory Tract
Cystic fibrosis (CF) is an autosomal recessive genetic disorder affecting individuals of northern European descent (117). CF is the most prevalent life-threatening inheritable disease in such individuals, with approximately one in 3,000 live births affected (118), and approximately 1,000 new diagnoses occurring annually in the United States of America (119). Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease.
Hundreds of unique mutations in CFTR are known to lead to CF, though the vast majority of cases (~85% in the United States) are due to the F508del mutation resulting in the loss of the phenylalanine residue at position 508 from CFTR (117, 119). While life expectancy for CF patients used to be in the teens, improved therapies have increased life expectancy to approximately age 40, and modeling predicts a child born with CF today will likely live to over age 50 (118-120).
Disease-causing mutations in CFTR can impair production of, function of, or trafficking to the cytoplasmic membrane by CFTR, an apical anion channel transporting chloride and
bicarbonate across the membrane (117, 121, 122). Mucosal epithelial cells are most affected by CFTR mutations, with impaired ion conductance leading to dehydration of the mucus layer, ATP channel disruption, organelle acidification, and dysregulation of intracellular vesicle transport (118, 123, 124). CF pathology is pronounced in several body sites: the intestines, where it causes the obstructive condition meconium ileus (125); the liver, where it blocks bile ducts and leads to focal biliary cirrhosis (126); the pancreas, promoting pancreatic insufficiency (127); and the respiratory tract, where disease manifestations cause the greatest number of deaths (119).
due to impaired CFTR function (or lack thereof), leading to the accumulation of dense, dehydrated mucus in the airways, which serves as a hospitable environment for invading opportunistic pathogens (117). Moreover, impaired innate immune defenses in the CF airways may contribute to infections (128), including dysfunctional defensins and antimicrobial peptides (possibly due to acidification of the mucus layer) (129, 130), as well as abrogated antimicrobial responses by neutrophils and biasing towards proinflammatory macrophages (131, 132). Canonical bacterial CF respiratory pathogens include Haemophilus influenzae, Staphylococcus aureus, P. aeruginosa, and members of the Bcc, though Stenotrophomonas maltophilia,
Achromobacter spp., and nontuberculous mycobacteria (NTM) are emerging CF pathogens (119, 133). Fungal pathogens can also infect CF patients, with Aspergillus fumigatus being the most concerning as it can cause invasive infections and allergic bronchopulmonary aspergillosis (134). Chronic infections caused by these pathogens promote perpetual inflammatory
responses, which damage the airways and lead to lung function decline and ultimately patient death (117, 118, 135).
Improvements in molecular techniques to identify microbial species have increased our understanding of polymicrobial communities in the CF airways. Although canonical pathogens can be the predominant organisms within these tissues (136-138), it is now clear that the CF respiratory tract harbors complex, dynamic microbial communities including nonpathogenic species (139). These techniques can also provide insight into the CF respiratory tract
Studies investigating such interactions are typically conducted ex vivo, as robust animal models for chronic CF infections have not been developed (145-147). P. aeruginosa and S. aureus are common focuses of sociomicrobiological investigations relevant to CF (148), and interactions between the two pathogens include competition (149, 150), alteration of antibiotic susceptibility (151, 152), and virulence potentiation (153-155). Continued development of CF animal models will make interbacterial interaction studies in the context of disease more accessible;
nevertheless such interactions have the potential to influence disease outcome in these patients (139, 148).
1.7 Pseudomonas aeruginosa
P. aeruginosa is a Gram-negative bacterium typically residing in aqueous environments
that notoriously causes a range of opportunistic infections in immunocompromised individuals (156). Given its metabolic versatility and regulatory networks coordinating adaptation to varying environments, P. aeruginosa causes infections in multiple body sites, including the eyes, ears, heart, urinary tract, skin, and respiratory tract (157). P. aeruginosa can cause chronic infections in the lungs and in burns and other wounds, and increasing rates of multidrug resistance limit antibiotic treatment options (158). As it is one of the most common pathogens causing healthcare-associated infections (159), there is a great need for research improving our knowledge of how P. aeruginosa causes infections and persists within human patients.
P. aeruginosa respiratory infections often occur in hospitalized patients intubated by ventilators, on which the bacterium can reside in biofilms, or in CF patients (156). Flagella and type IV pili mediate attachment to the respiratory epithelium and biofilm formation; both surface structures are required in infection models (160-162), though isolates from chronic infections often down-regulate flagellar gene expression or are non-flagellated (163). During acute
hypothesized the T3SS promotes P. aeruginosa wound infection by antagonizing wound healing and enhances neutrophil influx to infected lungs during pneumonia (165). Additional secreted virulence factors include the translation inhibitor exotoxin A (166, 167), several tissue-degrading proteases (156), lipases and phospholipases that degrade respiratory surfactant (162), oxidative stress-inducing pyocyanin (168), and iron-scavenging siderophores (169, 170). During the pro-inflammatory and tissue-destructive acute phase of infection, P. aeruginosa can breach the epithelial barrier and case fatal septicemia (157, 171). Quorum sensing, a density-dependent mechanism of intercellular communication and behavioral coordination, regulates virulence and biofilm formation by P. aeruginosa, and this communication is required during infection of animal models (172, 173).
If not cleared by the immune response or antibiotic treatment during acute infection, P. aeruginosa can transition to long term chronic infections of the respiratory tract where the bacteria typically reside within biofilms (156, 174). Despite the rich microbial communities in the CF airways, P. aeruginosa can be the predominant organism during chronic infections, making up over 90% of all bacterial cells (136-138). During the evolutionary process of host adaptation, mutations are selected for that promote biofilm formation and also reduce cytotoxicity and inflammatory potential (163, 175-178). Such adaptations include the loss of flagella and type IV pili, lack of T3SS activity, outer membrane alterations that decrease immune stimulation (179-181), conversion to a mucoidy state that may protect against immune responses (182), and inactivation of the lasR quorum sensing system (183). The Gac/Rsm global regulatory network controls transition from acute infection to chronic infection (184). GacS and GacA form a two-component system (GacS being the sensor kinase, and GacA being the response regulator), activating production of the small regulatory RNAs (sRNAs) RsmY and RsmZ when
T3SS, but induces expression of chronic infection-associated genes important for biofilm matrix production (pel and psl), siderophores, and the T6SS (157). GacSA phosphorelay is controlled by additional sensor kinases: RetS acts as a GacSA inhibitor (185), whereas LadS promotes GacSA phosphorelay (186).
Mutations in retS and gacS/gacA often determine the virulent lifestyle of P. aeruginosa within the CF respiratory tract. During early, acute phases of infection, RetS inhibits GacSA phosphorelay (184, 185); however, mutations can be selected for in retS that abrogate the activity of its protein product and allow for GacSA phosphorelay, and thus biofilm formation and other chronic infection behaviors (184, 187, 188). Given GacSA phosphorelay and RsmY/Z are required for production of T6SS proteins by P. aeruginosa, certain isolates from CF chronic infections can be T6SS-active (189), which may benefit this pathogen in the polymicrobial CF respiratory tract. After transitioning to a chronic infection lifestyle, often mediated by retS mutagenesis, subsequent mutations can be selected for in gacS/gacA (190-192), potentially abrogating GacSA phosphorelay and T6SS activity. In agreement with this, certain CF chronic infection isolates of P. aeruginosa are not T6SS-active (189). T6SS proteins of P. aeruginosa are immunogenic (189), so there may be a selective pressure to lose T6SS activity in later stages of infection.
P. aeruginosa is a model organism used to study T6S, as it has three separate T6SSs (the H1-, H2-, and H3-T6SS) (193). Both the H1- and H2-T6SSs mediate interbacterial
199). The phosphatase PppA dephosphorylates Fha1, a necessary step in the disassembly of the T6SS, promoting the formation of subsequent T6S apparatuses (199). TagQRST, PpkA, PppA, and Fha1 are all required for efficient T6SS activity and interbacterial competition (103, 199). It is hypothesized that the TagQRST system senses membrane perturbations and responds by activating PAO1 T6SS assembly, and membrane disruption may be due to T6SS attack from a neighboring competitor cell, thus promoting the T6SS-dueling behavior of PAO1 (103).
1.8 The Burkholderia cepacia Complex
The Bcc includes at least 17 species belonging to the Burkholderia genus that exhibit genetic diversity, but are phenotypically similar (200, 201). Bcc bacteria are found ubiquitously in the environment, often in soil and aqueous environments (201). In the soil, Bcc bacteria typically form beneficial symbioses with plants and prevent fungal infections (202), but can also be significant phytopathogens of onions and other commercial crops (203). The first identified Bcc bacterium originated from an onion infection, and was initially named Pseudomonas cepacia (204). 16S rRNA gene sequencing and DNA-DNA hybridization techniques have since determined Bcc species belong to the Burkholderia genus, not the Pseudomonas genus (205). Bcc bacteria have tremendous metabolic potential, including the ability to metabolize pollutants such as trichloroethylene, and thus have been used in bioremediation efforts (202, 206).
Despite their beneficial capabilities in promoting plant growth and reversing pollution, certain members of the Bcc cause serious opportunistic infections in immunocompromised patients (201). Humans most susceptible to Bcc infections are those with CF, but Bcc species are also problematic pathogens for individuals suffering from chronic granulomatous disease (CGD). CGD is a primary immune disorder that impairs the oxidative killing response of neutrophils, and thus Bcc pathogens can survive within the respiratory tracts of these
207). Similar to P. aeruginosa, Bcc pathogens can establish long-term infections in CF patients due to dysfunctional mucociliary transport and immune responses in the airways. The first description of Bcc bacteria as CF pathogens resulted from an epidemic during the early 1980s at the Hospital for Sick Children in Toronto (208). These patients showed more severe
symptoms and lung function decline than patients infected with P. aeruginosa (208), including necrotizing pneumonia, septicemia, and rapid death, a condition now referred to as “cepacia syndrome” (133, 201). Cepacia syndrome rarely occurs in patients infected by other canonical CF pathogens, and thus Bcc infections are of great concern among this patient population (209). Bcc pathogens can also transmit person-to-person and cause epidemics at CF treatment centers (210-213).
Compared to S. aureus and P. aeruginosa, Bcc infections are rare, occurring in 3-5% of CF patients within the United States (S. aureus and P. aeruginosa infect ~70% and ~45% of CF patients in the United States, respectively) (119). For unknown reasons, Bcc pathogens only infect older CF patients, typically teenagers and adults, with a median age at first infection of 20 years; conversely, S. aureus and P. aeruginosa are common in young patients, with S. aureus infecting 60-80% of patients 10 years old or younger, and a median age at first infection of 5 years for P. aeruginosa (119). Although Bcc pathogens can infect CF patients not culturing positive for P. aeruginosa, Bcc-P. aeruginosa co-infections are common, and in such cases, P. aeruginosa respiratory burdens are typically lower than in patients solely infected by P.
aeruginosa (208, 214). Like P. aeruginosa, Bcc pathogens can also be the predominant organisms in the CF respiratory tract during infections (137, 138). Notable Bcc pathogens are Burkholderia cenocepacia, B. multivorans, and B. dolosa, with B. cenocepacia and B.
Bcc bacteria exhibit a striking degree of intrinsic antibiotic resistance, making infections difficult to treat clinically (201). Resistance is often mediated by drug efflux pumps, decreased
OM permeability to antibiotics, and degradative enzymes including chromosomally-encoded b -lactamases (217-219). A notable example of antibiotic resistance within the Bcc is the ability of these bacteria to use penicillin as a sole carbon source (220). In addition to their antibiotic resistance potential, Bcc pathogens produce an array of classical virulence factors, though roles in pathogenesis have not been demonstrated for all (201). Bcc bacteria produce a
lipopolysaccharide (LPS) layer of the OM that is unique compared to other Gram-negative bacteria. Distinct chemical components of the LPS (specifically 4-amino-4-deoxyarabinose moieties attached to lipid A) inhibit the activity of antimicrobial peptides produced during the innate immune response (221, 222). Moreover, Bcc LPS is highly endotoxic and likely promotes inflammatory pathology during pneumonia and septicemia (223-225). Flagella are important for Bcc pathogen invasion of host cells, where these bacteria typically reside during infection (214), and flagellar mutants cause no overt disease in animal models of infection (226, 227). The B. cepacia epidemic strain marker, a genetic locus present in the ET-12 B. cenocepacia lineage, encodes a quorum sensing system that is required for virulence in a rat model of infection (228). Cable pili and a 22-kDa adhesin are produced by certain B. cenocepacia lineages and mediate attachment to the respiratory epithelium (229-231). Genes encoding a T3SS and one or more T6SSs are present in Bcc pathogens; disruptions in the T3SS-encoding locus and one T6SS-encoding locus attenuate virulence in murine models of infection (232, 233).
T6S has been studied in B. cenocepacia, mostly from the standpoint of its role in targeting host cells and promoting pathogenesis. By delivering the effector TecA (234), the B. cenocepacia T6SS inactivates macrophage Rho GTPases, disrupting their actin cytoskeleton, impairing phagocytosis, and inhibiting NADPH oxidase assembly onto vacuoles containing B. cenocepacia (235-238). TecA-induced cytoskeletal collapse activates the caspase-1
macrophages (234, 239, 240), exacerbating inflammation during murine infection. Pyroptosis activation by TecA is required for survival of B. cenocepacia-infected WT mice, though the role of T6SS-mediated inflammation in CF models in unknown. Recent bioinformatic analysis suggests this T6SS is prevalent among Bcc species (referred to as T6SS-1), though some species harbor additional T6SSs of unknown function (241). The T6SS-1 mediates modest interbacterial killing by B. cenocepacia strain H111 (241), but it remains unclear whether other Bcc pathogens, or additional T6SSs, have antibacterial functions. Given the ability of Bcc pathogens to establish infections in the polymicrobial CF respiratory tract, T6SS-mediated interbacterial competition may play an important role during initial colonization and/or prolonged infection.
1.9 Objectives of this Dissertation
CF is a polymicrobial disease characterized by complex, dynamic microbial communities in the airways that are often dominated by canonical pathogens. As interbacterial interactions likely occur in the CF respiratory tract and may influence pathogen colonization and overall disease, we hypothesize interbacterial competition provides pathogens advantages over other bacteria within the airways. Using Bcc bacteria as model organisms, we study CDI- and T6SS-mediated interbacterial competition through the following aims:
Aim 1: Characterize the potential CDI systems of B. dolosa and their ability to kill competitor bacteria.
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