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Published in final edited form as: Trends Microbiol. 2022 Apr 26;30(10):986–996. doi: 10.1016/j.tim.2022.03.009

Antibacterial Contact-dependent Proteins Secreted by Gram-negative Cystic Fibrosis Respiratory Pathogens

Cristian V Crisan a,b, Joanna B Goldberg a,b
PMCID: PMC9474641  NIHMSID: NIHMS1797228  PMID: 35487848

Abstract

Cystic fibrosis (CF) is a genetic disease that affects almost 100,000 people worldwide. CF patients suffer from chronic bacterial airway infections that are often polymicrobial and are the leading cause of mortality. Interactions between pathogens modulate expression of genes responsible for virulence and antibiotic resistance. One of the ways bacteria can interact is through contact-dependent systems, which secrete antibacterial proteins (effectors) that confer advantages to cells that harbor them. Here we highlight recent work that describes effectors used by Gram-negative CF pathogens to eliminate competitor bacteria. Understanding the mechanisms of secreted effectors may lead to novel insights into the ecology of bacteria that colonize respiratory tracts and could also pave the way for the design of new therapeutics.

Keywords: cystic fibrosis, bacterial pathogens, secretion systems, toxins

Different bacterial species can colonize the lungs of cystic fibrosis patients

Cystic fibrosis (CF) is a heritable condition that has a profoundly negative impact on the quality of life for affected individuals (please see “Glossary”) [1]. CF patients have a significantly shorter life expectancy than the general population and suffer from chronic bacterial lung infections [2]. These respiratory infections are the leading cause of CF mortality and are difficult to treat due to their ability to form biofilms, resist antibiotics, and evade immune responses [3].

Pseudomonas aeruginosa is found in the environment but is also a frequent pathogen detected in the lungs of CF patients [4]. The bacterium employs diverse mechanisms that allow it to resist antibiotic treatment and persist in lungs [4,5]. Following an acute infection phase, P. aeruginosa can alter expression of virulence genes to establish a chronic infection [4,5]. Chronic isolates form dense biofilms consisting of aggregated bacterial cells encased in a complex matrix of polysaccharides, proteins, DNA, and lipids [4]. Biofilms also allow P. aeruginosa cells to escape the immune system and evade phagocytosis [4]. Infections with the bacterium are the leading cause of mortality among CF patients and approximately 80% of all patients suffer from chronic infection by the age of 20 [6,7].

P. aeruginosa has been considered the main CF airway pathogen but other Gram-negative bacteria are important contributors to disease progression. The Burkholderia cepacia complex includes a group of closely-related bacterial species that infect CF patients [8]. Isolates are often resistant to multiple antibiotics, encode diverse virulence factors, and can lead to a rapid decline in lung function [8]. Stenotrophomonas maltophilia is an emerging multidrug-resistant pathogen that has increased in prevalence among CF patient in recent years [9]. The bacterium is a natural inhabitant of soil and wet ecosystems but is often acquired from hospitals, where it survives on medical equipment and cleaning solutions [9]. S. maltophilia CF airway infections may be associated with increased symptom severity, a pronounced immunogenic response, decreased lung function, and higher mortality [9]. Other Gram-negative pathogens, including Haemophilus influenzae and Achromobacter xylosoxidans, also infect lungs and may negatively impact disease outcomes [10,11]. Staphylococcus aureus is the most prevalent Gram-positive bacterial species isolated from CF samples and is frequently detected in younger patients [3].

Microbial interactions modulate important traits in CF pathogens

Recently, polymicrobial respiratory infections and the bacterial interactions between these infecting species have been recognized as critical health factor determinants in CF lung disease [3]. Different CF pathogens can concomitantly colonize respiratory tracts and polymicrobial infections are correlated with poor clinical outcomes [3]. Diverse polymicrobial interactions that modulate virulence, alter physiology, influence persistence, and suppress immune responses have been reported in CF pathogens. For example, P. aeruginosa produces virulent diffusible secondary metabolites that damage S. aureus cells, promote dispersal from biofilms, and modulate resistance to antibiotics [12]. The secreted P. aeruginosa exopolysaccharide alginate confers protection to S. aureus against cell death due to P. aeruginosa aggression and supports clinical reports that detect both bacterial species in CF patient lungs [13].

The spatial ecology in lungs can limit the ability of pathogens to engage in interactions by creating physical distances between cells [14]. CF pathogens are often found in aggregates and multiple species may compete for space and resources [15]. Recently, P. aeruginosa has been observed to arrange into highly ordered arrays where cells come into direct contact when cultured in CF growth medium [16]. Furthermore, polymicrobial interactions affect the ecological distribution of CF bacteria. Both Burkholderia cenocepacia and S. maltophilia influence the spatial organization of P. aeruginosa by secreting unsaturated fatty acids, while S. maltophilia can further modulate P. aeruginosa biofilm structures by producing a diffusible signal factor [17,18].

Polymicrobial interactions between Gram-negative bacteria may be mediated by contact-dependent proteins that have antibacterial properties and are transferred between adjacent cells (Fig. 1) [19–21]. A common feature among those systems is the presence of immunity proteins, which protect self and kin cells from intoxication. Contact-dependent antibacterial proteins could play important roles in conferring competitive advantages to bacteria that harbor them. They may allow invader strains to eliminate established species or confer protective mechanisms to resident cells against foreign bacteria. In this review, we highlight recent findings that describe antibacterial proteins secreted by P. aeruginosa, species of the B. cepacia complex, and S. maltophilia to inflict harm upon other bacterial species in a contact-dependent manner. We focus our discussion on antibacterial contact-dependent proteins and their molecular mechanisms used in both intra- and inter-species competition. Since antimicrobial resistance among CF pathogens is widespread, strategies to eliminate resistant strains are urgently needed and contact-dependent antibacterial proteins might provide insights into the development of novel therapeutics.

Figure 1. CF pathogens employ contact-dependent inhibition systems, type VI secretion systems and type IV secretion systems to eliminate target bacteria.

Figure 1.

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A) Contact-dependent inhibition (CDI) genes have been identified in both Pseudomonas aeruginosa and Burkholderia cepacia complex species. CdiA proteins are long, filamentous and bind receptor proteins on target cells to deliver toxic effectors. CdiB proteins form the channel through which CdiA proteins are excreted. CDI systems are effective at intoxicating the same or closely related bacterial species. B) The Type VI Secretion System (T6SS) is a large macromolecular structure that delivers toxins into adjacent cells using a contraction process. A multiprotein membrane complex and baseplate structure facilitate the excretion of an Hcp tubule that is capped by a VgrG/PAAR protein tip. C) Type IV Secretion Systems (T4SS) exhibit large structural diversity but generally require an inner and an outer membrane complex. Pilins facilitate contact with target cells and might help deliver toxic effectors.

Contact-dependent inhibition systems mediate competition between the same or closely related species

Contact-dependent inhibition (CDI) systems were first described in Escherichia coli but bacteria from the Burkholderia and Pseudomonas genera have been shown to employ CDI toxins to eliminate competitor cells from the same or related species (Fig. 1) [19,22–24]. CDI systems are also referred to as Type Vb Secretion and require three core genes: cdiA, cdiB and cdiI [19,25]. CdiA proteins are heterogenous, large, and form filamentous structures [26]. Their conserved N-terminal regions encode a Sec-dependent signal for delivery to the periplasm of inhibitor cells and filamentous hemagglutinin (FHA) repeats that facilitate binding to receptors on target cells [27]. CdiA proteins are thought to extend from the cell surface and interact with receptors on target cells via receptor-binding domains (RBD) [27,28]. Multiple outer membrane proteins have been identified as CdiA receptors on E. coli cells, including the β-barrel assembly machine BamA protein, the Tsx outer membrane nucleoside transporter, and the OmpC and OmpF porins [28–31]. The DppB and DppC peptide transport system components were recently identified as CdiA receptors in P. aeruginosa [32]. C-terminal regions of CdiA proteins contain varied toxin domains with diverse antibacterial activities [22]. CdiB proteins assemble into β-barrel structures with transmembrane strands that insert in the membranes of inhibitor cells and facilitate secretion of CdiA [26,33]. Conformational changes induce the opening of the CdiB pore to allow passage of CdiA proteins from the periplasm to the cell surface [33]. Immunity proteins (CdiI) bind and neutralize specific CdiA toxins and prevent intoxication of self and kin cells [19,34].

The CDI system in Burkholderia was first described in the biodefense pathogen Burkholderia pseudomallei, which encodes CdiA proteins with C-terminal domains that cleave tRNA and inhibit target cells [35] (Fig. 2). Distinct B. pseudomallei CdiA proteins target specific tRNA molecules [35]. In some Burkholderia species, CDI systems are encoded by operons designated as bcpAIOB, where BcpA proteins contain C-terminal toxic domains, BcpB form membrane β-barrel structures and BcpI confer immunity, respectively [36]. The function of BcpO is unknown. Burkholderia thailandensis, a soil species related to B. pseudomallei, contains functional bcpAIOB gene operons that are used to eliminate target related bacterial cells, alter biofilm structures and gain ecological advantages [36]. Two Burkholderia species that infect CF patients, Burkholderia multivorans and Burkholderia dolosa, employ CDI systems with potent antimicrobial properties [23,37]. Both species encode putative BcpA proteins, which likely intoxicate strains from the same or related strains [23,37]. It is intriguing to speculate that these isolates might use CDI systems to establish themselves as the dominant strains in CF patients. P. aeruginosa isolates (including from CF patients) also encode multiple CDI operons (Fig. 1) [32,38,39]. Similar to other organisms, the N-terminal region of predicted CdiA proteins from P. aeruginosa is highly conserved, while the C-terminal region contains diverse domains with putative toxic functions like nucleases, peptidases and deaminases [24,38] (Fig. 2). CdiA proteins confer competitive advantages to P. aeruginosa against related bacterial cells, exhibit toxicity when expressed inside E. coli, and contribute to biofilm formation [24]. The recently described P. aeruginosa Cdi1APABL017 exhibits tRNAse activity and is required for intra-species competition [32]. Cdi1APABL017 is remarkable because it provides the first in vivo evidence of a direct role for CDI in virulence towards mice [32].

Figure 2. Proteins delivered in a contact-dependent manner by CF pathogens possess diverse molecular mechanisms to damage target bacterial cells.

Figure 2.

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Antibacterial proteins secreted by CDI, T6SS and T4SS of CF pathogens possess activity in both the cytoplasm and cellular envelope. Lipases, pore forming toxins and metallopeptidase damage the membrane, while amidases and glycosyl hydrolases disrupt the peptidoglycan cell wall. Cytoplasmic nucleotides (NAD(+) and (p)ppApp) and nucleic acids (DNA and tRNA) are also targeted by contact-dependent toxins.

The Type VI Secretion System translocates a cocktail of toxins into adjacent cells

The Type VI Secretion System (T6SS) is an antibacterial weapon used by many important Gram-negative human pathogens to deliver toxic proteins into adjacent cells (Fig. 1) [20,40–42]. Components of the apparatus share homology to bacteriophage proteins and resemble a harpoon decorated with toxins [43]. The T6SS is assembled and anchored on a multiprotein membrane complex that spans the periplasmic space [44]. A baseplate structure is localized at the bottom of the membrane complex via several protein-protein interactions [45]. Hexamers of Hcps (Hemolysin Coregulated Proteins) form a cylindrical tube that can extend across an entire bacterial cell [20]. Some Hcp proteins bind T6SS effectors and facilitate their secretion [46]. At the distal end, VgrGs (valine-glycine repeat protein G) assemble to form the tip of the apparatus and help load T6SS effectors onto the apparatus [47,48]. Several VgrGs also contain effector domains that diversify the toxin repertoire delivered into target cells [49,50]. PAAR (proline-alanine-alanine-arginine) repeat proteins bind VgrGs and are thought to “sharpen” the tip and help deliver additional toxins [51].

The secretion process is facilitated by outer sheath proteins TssB and TssC that surround the Hcp tubule [52]. TssB and TssC coordinate a contraction and extension mechanism that propels the Hcp tube, VgrG tip and loaded effectors outside the cell. The TssB/TssC complex is not secreted, but is instead recycled by ClpV, an ATP-dependent AAA+ protein [53]. Depending on the depth of penetration, effectors may access both the periplasmic space and cytoplasm of target Gram-negative bacteria. An important feature of the T6SS is the presence of immunity proteins that are generally encoded near toxin genes and prevent self-intoxication by neutralizing effectors [54]. P. aeruginosa was among the first bacterial species in which the T6SS was discovered [20]. In P. aeruginosa, T6SS antibacterial effectors can be divided into three classes based on the cellular location of their toxic activity: effectors that damage the membrane, effectors that disrupt the cell wall, and effectors that act in the cytoplasm (Fig. 2).

Lipases represent a diverse family of T6SS effectors that hydrolyze membrane lipids of target bacteria. T6SS lipase effectors (Tle) can be classified into 5 groups [55]. Tle1 adopts a canonical α/β-hydrolase fold and requires the Ser-Asp-His catalytic triad for its hydrolyzing activity [56]. Tle3 is toxic when expressed in the E. coli periplasm and is neutralized by its cognate Tli3 immunity protein [57]. Tle4 also adopts a α/β-hydrolase fold, is toxic when delivered to the periplasm, and is inhibited by the Tli4 cognate immunity protein [58,59]. Tle5a and Tle5b possess HxKxxxxD motifs and the phospholipase D activity of Tle5a was experimentally validated [55,60]. Unlike phospholipases that actively degrade lipids, Tse4 is a relatively small protein that is predicted to form ion-selective pores in target cell membranes when delivered by the P. aeruginosa T6SS [61]. The VgrG2b tip-forming protein contains a metallopeptidase C-terminal domain that causes morphological defects in target cells, depletes lipoproteins and perturbs cell division [62].

The bacterial cell wall is composed of peptidoglycan, a polymeric structure with linear strands of alternating N-acetylglucosamine and N-acetylmuramic acid monomers crosslinked by peptides [63]. P. aeruginosa T6SS effectors that degrade peptidoglycan exhibit either amidase activity (named Tae, type VI amidase effectors) or glycoside hydrolase activity (named Tge, type VI glycoside hydrolase effectors) [64,65]. Both P. aeruginosa Tae1 and AmpDh3 effectors have amidase activity, and Tae1 specifically cleaves bonds between γ-d-glutamic acid and meso-diaminopimelic acid molecules [64–67]. The glycoside hydrolase Tge1 has β-(1,4)-N-acetylmuramidase catalytic activity and is used by P. aeruginosa to intoxicate target bacteria in a T6SS-depedent manner when delivered to the periplasm [64]. Tse5 contains a C-terminal domain that is toxic when expressed in the periplasm and an Rhs (rearrangement hotspot) N-terminal domain proposed to facilitate its excretion [68].

Several P. aeruginosa T6SS effectors display toxic activity in the bacterial cytoplasm. Tse2 is likely an NAD-dependent bacteriostatic toxin that is inhibited by the cognate Tsi2 immunity protein [54,66]. Tse6 cleaves NAD(P)+ molecules and requires the EF-Tu elongation factor for its delivery into the target cells’ cytoplasm [69]. The P. aeruginosa PAK Tse7 effector contains a C-terminal Tox-GHH2 nuclease domain and degrades DNA [70]. Similarly, TseT encodes a Tox-REase domain predicted to intoxicate target bacteria [71]. Ahmad et al. recently reported that the Tas1 toxin from the virulent P. aeruginosa PA14 strain is a highly efficient (p)ppApp synthase [72]. Tas1 depletes ATP and ADP molecules and alters essential cellular pathways involved in energy generation, nucleotide metabolism and cell envelope synthesis [72]. Tas1 thus provided the first experimental evidence for the unexpected metabolic role played by a (p)ppApp synthase in bacterial cells [72]. Tse8 is another recently described cytosolic T6SS toxin that interacts with the multimeric transamidosome ribonucleoprotein and impairs protein synthesis [73].

While P. aeruginosa is a potent bacterial cell killer, it can become itself the target of other bacterial pathogens, including species from the B. cepacia complex [74] (Fig. 1). The organization and distribution of T6SS genes in the Burkholderia genus is diverse, with some species harboring multiple distinct T6SS clusters. B. thailandensis encodes effectors that act on the bacterial cell wall (with amidase activity) or cell membrane (with Tle1-like lipase activity) [55,75]. T6SS toxins with cytoplasmic activity have also been identified in Burkholderia species. TseTBg effectors are encoded by Burkholderia gladioli and exhibit both DNAse and RNAse activity [76]. Moraes et al. found that B. cenocepacia uses the T6SS to translocate DddA, a cytosine deaminase toxin, into target cells [77]. Some bacterial species resist DddA-mediated toxicity and acquire mutations that can have potentially beneficial consequences like resistance to antibiotics [77]. DddA is a remarkable example of how the activity of a contact-dependent toxin can be exploited to perform a different function that its original one [78,79]. Mok et al. split DddA in two halves which, when fused to TALE (transcription activator-like effector) proteins, enabled efficient editing of mitochondrial DNA in human cells [78]. A subsequent study by Lee et al. showed DddA-TALE fusions could be used in vivo to induce mutations to mitochondrial DNA in mice [79].

The Type IV Secretion System is a versatile protein export machine that also delivers antimicrobial proteins

The Type IV Secretion System (T4SS) is a contact-dependent macromolecular structure used by both Gram-negative and Gram-positive bacterial species [80]. Many pathogens use the T4SS for horizontal gene transfer, but the apparatus also excretes virulence factors that damage eukaryotic cells and modulate immune responses [80]. T4SSs exhibit a wide diversity and are categorized into two groups: Type IVA and Type IVB. Here we focus on the Type IVA system since it has been recently experimentally validated to deliver antibacterial proteins [81,82].

The Type IVA system requires approximately one dozen core components (named VirB1 – VirB11 and VirD4) and can structurally be divided into three multiprotein subunits: the inner membrane complex, the outer membrane complex and the pilus structure [83]. VirB1 is a periplasmic protein that is thought to locally lyse peptidoglycan and create the necessary space for T4SS assembly [84]. The inner membrane complex includes several structural proteins with transmembrane helices (VirB3, VirB6 and VirB8) and ATPase proteins (VirB4, VirD4 and VirB11) [83,85–88]. VirD4 plays important roles in transferring substrates to the T4SS for the excretion process [89]. The VirB7 lipoprotein, VirB9 protein and the C-terminal domain of VirB10 form a stable, core complex in the outer membrane that is connected to the inner membrane complex by a cylindrical linker [90–92]. The T4SS pilus structure is primarily composed of the VirB2 protein, which polymerizes to adopt a filamentous structure topped at the distal end by VirB5 proteins [93].

The T4SS was first identified as an antibacterial weapon in the plant pathogen Xanthomonas citri [81,82]. The opportunistic CF pathogen S. maltophilia, which belongs to the Xanthomonadaceae family, also contains functional virB genes [21,94]. The T4SS of S. maltophilia strain K279a was recently shown to be utilized in competition with other bacteria, including P. aeruginosa isolates from CF patients (Fig. 1) [21,94,95]. Multiple putative T4SS toxins have been identified with predicted activities such as lipase, lysozyme-like, and nuclease [94,95]. Smlt3024 is important because it is the first S. maltophilia T4SS effector for which experimental evidence exists to demonstrate its antibacterial potential. Although its biochemical function is not immediately apparent, Smlt3024 is toxic when delivered to the E. coli periplasm, shares homology to proteins with RTX (repeats in toxin) motifs, and is inhibited by the adjacently encoded Smlt3025 immunity protein [94]. Nas et al. recently demonstrated that the S. maltophilia proteins TfcA (predicted to be a lipase) and TfcB (predicted to have lysozyme-like activity) contribute to T4SS-depedent competition against clinical P. aeruginosa CF isolates [95].

Concluding remarks and future directions

Contact-dependent antibacterial proteins are versatile weapons against competitor cells. Different effectors target specific structures in all cellular compartments (Fig. 2). Although many putative antimicrobial proteins secreted in a contact-dependent manner have been identified, in some cases experimental evidence to demonstrate their predicted toxic mechanisms is lacking. It is also unclear how environmental cues and host factors might influence the expression and toxic activity of contact-dependent effectors used by CF pathogens. Most studies have examined antimicrobial proteins under standard laboratory conditions. However, less is known about their importance during CF infections. Recent advances have uncovered unexpected activities that have led to the engineering of antibacterial proteins for new functions such as novel DNA editing tools, suggesting these proteins are potential untapped resources [78,79].

Discoveries about the roles of bacterial contact-dependent protein secretion in other model systems could provide important insights into our understanding of competition among CF pathogens in respiratory tracts. For example, antibacterial proteins delivered by the T6SS are used by enteric pathogens to displace other species and increase virulence. Vibrio cholerae, the pathogen responsible for cholera disease, employs T6SS antibacterial toxins to eliminate E. coli commensal strains in vivo and colonize intestines [96]. Fast et al. used a Drosophila model system to demonstrate that T6SS-mediated interactions of V. cholerae with symbiont bacterial species contribute to cholera-like symptoms [97]. Furthermore, T6SS-mediated killing of commensals and subsequent establishment in the guts of mice was also observed for Salmonella Typhimurium [98].

Studies have speculated that CF pathogens might use CDI and T6SS to gain competitive and ecological advantages in the respiratory tract (Fig. 3) [23,60,74]. In a similar manner to gut microbiome species, commensal bacteria from upper respiratory tracts may confer protection against opportunistic pathogens [99]. P. aeruginosa persists in upper respiratory tracts of mice and migrates towards lungs to establish chronic infections [100]. We propose that in the context of CF, opportunistic pathogens may also secrete antibacterial proteins to displace commensal species of the upper respiratory tract to establish initial infections. Secretion systems could then provide further benefits for pathogens while inside the lungs of CF patients.

Figure 3. Contact-dependent antibacterial proteins could contribute to the persistence of CF pathogens in both the external environment and inside lungs.

Figure 3.

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P. aeruginosa, species from the B. cepacia complex and S. maltophilia are often isolated from environmental sources and hospital settings where competition for space and resources is critical for survival. The ability to eliminate target cells using contact-dependent antimicrobial proteins could confer significant competitive advantages to killer cells and allow them to survive in diverse environments. Subsequently, those bacterial pathogens could infect CF patients and use contact-dependent mechanisms to eliminate other bacteria and establish chronic infections.

CDI and T6SS systems contribute to the ability of P. aeruginosa to cause in vivo infections and genes coding for CDI and T6SS systems have been detected in the genomes of bacterial isolates from CF patients [32,39,70,74]. La Rosa et al. have recently predicted that P. aeruginosa chronic isolates have higher T6SS gene expression compared to acute strains, suggesting the apparatus might function as a defensive weapon for established species against foreign invaders [101]. The hypothesis that chronic P. aeruginosa strains employ active T6SS is supported by the finding that Hcp, a known protein marker for T6SS secretion, was detected in sputum samples of patients chronically infected with P. aeruginosa [20]. Antibodies against Hcp were also present in patients with chronic infections, suggesting that an immunologic response against T6SS substrates exists [20]. By contrast, Perault et. al have shown that B. cenocepacia utilizes the T6SS to eliminate P. aeruginosa strains isolated from older patients [74]. P. aeruginosa strains from younger patients display T6SS-mediated activity but isolates from older patients have loss-of-function T6SS mutations that render them susceptible to in vitro killing by B. cenocepacia [74]. Taking all these findings into consideration, it is possible that a “bacterial warzone” exists inside the lungs of CF patients and bacteria that possess weapons like the T6SS, CDI or T4SS have survival advantages. Intriguingly, some T6SS and CDI effectors that are toxic to prokaryotic cells also target eukaryotic cells [32,57,60]. Furthermore, internal and external factors that allow bacteria to resist contact-dependent killing independently of immunity proteins have been recently identified [102–104].

Since many CF pathogens are broadly distributed in external environments, CDI, the T6SS and the T4SS could contribute to the survival and subsequent acquisition of pathogens by susceptible individuals (Fig. 3). The spatial organization of polymicrobial infections can affect the ability of pathogens to come into direct contact with other bacterial cells. Additional work is necessary to experimentally validate that contact-dependent antibacterial proteins facilitate pathogen acquisition and contribute to chronic infection. However, invasion events mediated by contact killing during which foreign bacteria rapidly eliminate established species might be difficult to observe and study in patients.

Considering recent findings that the T4SS contributes to antibacterial killing, it is interesting to inquire if evolutionary pressures drive a bacterium to choose between using the T6SS or the T4SS to eliminate diverse competitor cells. It is possible that complex factors like the energy required to deliver proteins, the ability of toxins to eliminate specific competitor bacteria, and external conditions that mediate toxic activity play roles in determining which secretion system is used as the “weapon of choice”. Future work will determine if other contact-dependent secretion systems of CF pathogens might also deliver antimicrobial proteins (see ‘Outstanding Questions‘).

Outstanding Questions.

  • Do contact-dependent interactions between cystic fibrosis (CF) pathogens occur frequently in lungs? If they do, are they transient and do they impact the microbial spatial ecology?

  • How are secretion systems (or the effectors) regulated during CF lung infections?

  • How do secretion systems respond to fluctuations in environmental conditions?

  • What are the cues used by CF pathogens in the lungs to alter expression of secretion system proteins?

  • Are there evolutionary or competitive advantages to choosing between one or another secretion system to deliver antibacterial proteins?

  • Are there other, undiscovered secretion systems that also deliver antibacterial proteins in CF pathogens?

  • Do other emerging CF pathogens use contact-dependent secretion systems to eliminate bacterial cells?

  • Do antimicrobial proteins secreted by contact-dependent systems contribute to the survival of pathogens in external environments and subsequent acquisition by patients?

  • How do external conditions influence the efficacy of secreted toxins?

  • How do CF pathogens evolve resistance against contact-dependent antibacterial proteins?

  • Can secreted proteins be repurposed and engineered as therapeutics?

Since many CF pathogens display resistance to a wide range of antibiotics, secreted proteins might serve as novel scaffolds for potential treatments in the future. Additionally, non-pathogenic bacterial strains that eliminate CF pathogens in a contact-dependent manner could be engineered as potential probiotic treatments and efforts to make such strains are already underway [105]. Understanding the mechanisms of contact-dependent antibacterial toxins is crucial for the design of probiotic strains that employ them to ensure that pathogenic strains can be efficiently targeted and eliminated. Such probiotic strains could be fine-tuned to exhibit only local bactericidal activity and have fewer systemic side effects compared to diffusible molecules. Future studies are required to better understand the regulation of contact-dependent secretion systems, to explore the repertoire of delivered toxins, and to investigate the roles that external signals play in determining outcomes of antimicrobial protein secretion.

Highlights.

  • Cystic fibrosis (CF) is an inherited disease that negatively impacts the lives of almost 100,000 people worldwide; chronic polymicrobial respiratory infections are critical to patient outcome

  • Opportunistic bacterial pathogens like Pseudomonas aeruginosa, members of the Burkholderia cepacia complex and Stenotrophomonas maltophilia can be co-isolated from the lungs of CF patients

  • Contact-dependent inhibition systems may allow CF pathogens to eliminate bacteria from the same or closely related species

  • P. aeruginosa and Burkholderia species can deliver antibacterial proteins via a Type VI Secretion System to alter the target bacterial cells’ metabolism and degrade the cell envelope

  • S. maltophilia can employ a Type IV Secretion System to translocate antibacterial proteins that contribute to the pathogen’s competitive fitness

Acknowledgements

This work was supported by grants from the Cystic Fibrosis Foundation (GOLDBE19P0 and WHITEL20A0) and the NIH (R21-AI48847). We thank members of the Goldberg lab, especially Dr. Rebecca Duncan, Rachel Done, Justin Luu, Ashley Alexander, and Vishnu Raghuram for critically reading the manuscript and providing suggestions. We also thank Dr. Sheyda Azimi and Madeline Mei from the Diggle lab at Georgia Institute of Technology for their helpful comments.

Glossary

BamA

protein involved in the assembly of other outer membrane proteins that serves as a receptor for contact-dependent inhibition in E. coli

Bcp

proteins involved in the Burkholderia contact-dependent inhibition systems

CDI

contact-dependent inhibition, a bacterial secretion system that transfers proteins from “inhibitor” bacterial cells to adjacent “target” cells

CdiA

large filamentous protein involved in contact-dependent inhibition that usually encodes C-terminal toxic domains

CdiB

β-barrel proteins that facilitate CdiA export during contact-dependent inhibition

CidI

immunity proteins that neutralize CdiA toxins

CF

cystic fibrosis, a genetic disease that causes mucus accumulation in organs like lungs, pancreas and intestines

ClpV

ATPase that recycles components of the Type VI Secretion System following a secretion event

DppB/DppC

peptide transport system components that serve as receptors for CdiA in P. aeruginosa

Effector

a secreted bacterial molecule that confers some competitive or nutritional advantage to the cell that harbors it

Hcp

Hemolysin Coregulated Proteins that form the hexameric inner tube of Type VI Secretion Systems

Immunity Protein

protein involved in neutralizing the toxicity of an antibacterial effector

Omps

outer membrane proteins involved in protein secretion and nutrient uptake; OmpC and OmpF are receptors for contact-dependent inhibition in E. coli

PAAR

proline-alanine-alanine-arginine repeat proteins that form the tip of the Type VI Secretion System apparatus and may deliver toxic effectors

(p)ppApp

nucleotide alarmone that regulates many bacterial metabolic processes like sugar metabolism, cell envelope synthesis and nucleoside metabolism

T4SS

Type IV Secretion System, a bacterial secretion system involved in multiple processes like DNA conjugation, transformation, and protein export

T6SS

Type VI Secretion System, a contact-dependent bacterial secretion system that exports proteins and mediates processes like virulence and bacterial competition

Tas

Type VI secretion effector (p)ppApp synthetase; toxic protein secreted by the Type VI Secretion System that efficiently catalyzes synthesis of (p)ppApp in target cells

Tae

Type VI amidase effectors; toxic proteins secreted by the Type VI Secretion System that degrade peptidoglycan in target cells

Tge

Type VI glycoside hydrolase effectors; toxic proteins that degrade peptidoglycan in target cells

Tse

Type VI effectors; toxic proteins secreted by the Type VI Secretion System with diverse antibacterial mechanisms

Tle

Type VI lipase effectors; toxic proteins that degrade the lipid membranes of target cells

TssB/TssC

proteins that form the contractile sheath of the Type VI Secretion System

Tsx

integral outer membrane protein that forms a channel for nucleosides and serves as a receptor for contact-dependent inhibition in E. coli

VgrG

valine-glycine repeat protein G, forms the trimeric tip of the Type VI Secretion System apparatus and aids in toxin delivery

VirB/D

proteins involved in the structure and/or function of the Type IV Secretion System

Footnotes

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