The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short form (TssAS) and a long form (TssAL). While TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for some time or fire immediately after sheath extension. We propose that this diversity in firing dynamics could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.
Pseudomonas aeruginosa is a significant nosocomial pathogen and associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN, and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis and catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), that are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress, and responds to both oxidants by upregulating expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated) there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type III secretion system, sulphur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role. IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress, and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type III secretion system.
Pseudomonas aeruginosa is a significant nosocomial pathogen and associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite the host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and survives attack from HOCl and HOSCN, and the contribution of such responses to its success as a CF pathogen. We investigated the P. aeruginosa response to these oxidants by screening 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators involved in antibiotic resistance, methionine biosynthesis and catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), that were required for HOCl survival. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress, and responds to both oxidants by upregulating expression of a putative peroxiredoxin, rclX (PA14_07355). While there was specificity in the transcriptional response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated) there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type III secretion system (T3SS), sulphur and taurine transport, and
20 21 2 These authors have contributed equally to this work. 22 23 24 KEYWORDS 25 26 Type VI secretion system, interbacterial competition, contact-dependent killing, toxin 27 delivery, TssA, TagB, TagA, sheath stabilization, sheath contraction, Pseudomonas. 28 29 30 ABSTRACT 31 32The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily 33 involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the 34 assembly of the T6SS sheath structure, the contraction of which propels a payload of effector 35proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected 36 diversity and exist in two major forms, a short (TssAS) and a long (TssAL) TssA. Whilst 37TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended 38sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. 39Here we discover a novel class of structural components that interact with short TssA 40proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the 41 baseplate. We demonstrate that the presence of these components is important for full sheath 42 extension and optimal firing. Moreover, we show that the pairing of each form of TssA with 43 a different class of sheath stabilization proteins results in T6SS apparatuses that either reside 44in the cell for a while or fire immediately after sheath extension, thus giving rise to different 45 aggression behaviors. We propose that this functional diversity could contribute to the 46 specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches. 47 48 49Bacteria live in complex polymicrobial communities that are shaped by interspecies 50 cooperation and competition. As resources are limited, antagonistic strategies are a major 51 driver of survival and success for bacterial populations. One of the most elaborate bacterial 52weapons is the type VI secretion system (T6SS), which not only promotes inter-bacterial and 53inter-kingdom competition (1-3), but is also involved in the interaction of bacteria with their 54
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