Rhs NADase effectors and their immunity proteins are exchangeable mediators of inter-bacterial competition in Serratia.

Many bacterial species use Type VI secretion systems (T6SSs) to deliver anti-bacterial effector proteins into neighbouring bacterial cells, representing an important mechanism of inter-bacterial competition. Specific immunity proteins protect bacteria from the toxic action of their own effectors, whilst orphan immunity proteins without a cognate effector may provide protection against incoming effectors from non-self competitors. T6SS-dependent Rhs effectors contain a variable C-terminal toxin domain (CT), with the cognate immunity protein encoded immediately downstream of the effector. Here, we demonstrate that Rhs1 effectors from two strains of Serratia marcescens, the model strain Db10 and clinical isolate SJC1036, possess distinct CTs which both display NAD(P)+ glycohydrolase activity but belong to different subgroups of NADase from each other and other T6SS-associated NADases. Comparative structural analysis identifies conserved functions required for NADase activity and reveals that unrelated NADase immunity proteins utilise a common mechanism of effector inhibition. By replicating a natural recombination event, we show successful functional exchange of CTs and demonstrate that Db10 encodes an orphan immunity protein which provides protection against T6SS-delivered SJC1036 NADase. Our findings highlight the flexible use of Rhs effectors and orphan immunity proteins during inter-strain competition and the repeated adoption of NADase toxins as weapons against bacterial cells.

Impact of Sociodemographic Disparities on Language Outcomes After Cochlear Implantation in a Diverse Pediatric Cohort.

OBJECTIVE: We examined how sociodemographic and audiologic factors affect receptive and expressive language outcomes in children with cochlear implantation. STUDY DESIGN: Retrospective cohort study. SETTING: A hearing loss (HL) clinic at a tertiary center. METHODS: Sociodemographic variables, HL characteristics, age at implantation, and receptive language scores (Preschool Language Scale and the Clinical Evaluation of Language Fundamentals) were collected from patients with congenital HL who received their first implant by 4 years old after January 1, 2007. t Tests, linear regression, Mann-Whitney, Cohen's d, and mediation analysis were used for descriptive statistics and hypothesis testing. RESULTS: Among 79 patients, 42 (53%) were females, 44 (56%) under-represented minorities, and 56 (71%) had public insurance. At least 1 year after implantation, the median receptive language score was 69 (range 50-117). Females (p = .005), having private insurance (p = .00001), having a Cochlear Implant Profile score below 4 (p = .0001), and receiving their implant at or before 12 months of age (p = .0009) were significantly associated with improved receptive language outcomes. Insurance type had a significant effect on receptive language outcomes, independent from age at first implantation (total effect: coef = -13.00, p = .02; direct effect: coef = -12.26, p = .03; indirect effect: coef = -0.75, p = .47). Sociodemographic variables had large effect sizes, with the Cochlear Implant Profile score having the largest effect size (d = 1.3). CONCLUSION: Sociodemographic factors have a large impact on receptive language outcomes. Public insurance is associated with worse receptive language, not mediated by later age at implantation, suggesting that other factors primarily impact language outcomes in publicly insured children with cochlear implants.

The accessory protein TagV is required for full Type VI secretion system activity in Serratia marcescens.

The bacterial Type VI secretion system (T6SS) is a dynamic macromolecular structure that promotes inter- and intra-species competition through the delivery of toxic effector proteins into neighbouring cells. The T6SS contains 14 well-characterised core proteins necessary for effector delivery (TssA-M, PAAR). In this study, we have identified a novel accessory component required for optimal T6SS activity in the opportunistic pathogen Serratia marcescens, which we name TagV. Deletion of tagV, which encodes an outer membrane lipoprotein, caused a reduction in the T6SS-dependent antibacterial activity of S. marcescens Db10. Mutants of S. marcescens lacking the core component TssJ, a distinct outer membrane lipoprotein previously considered essential for T6SS firing, retained a modest T6SS activity that could be abolished through deletion of tagV. TagV did not interact with the T6SS membrane complex proteins TssL or TssM, but is proposed to bind to peptidoglycan, indicating that the mechanism by which TagV promotes T6SS firing differs from that of TssJ. Homologues of tagV were identified in several other bacterial genera, suggesting that the accessory function of TagV is not restricted to S. marcescens. Together, our findings support the existence of a second, TssJ-independent mechanism for T6SS firing that is dependent upon the activity of TagV proteins.

The structure of a tautomerase superfamily member linked to the type VI secretion system of Acinetobacter baumannii.

Bacteria exploit specialized secretion systems to assist in competition for resources, in collaboration and in communication. Here, a protocol for the recombinant production, purification and crystallization of a protein linked to the Acinetobacter baumannii type VI secretion system is provided. A high-resolution structure of this trimeric protein is reported, revealing the characteristic dual ß-α-ß subunit fold typical of longer subunit members of the tautomerase superfamily. The protein does not appear to be toxic to bacteria or yeast under the conditions tested. The possible biological role of this protein is discussed.

The genus Serratia revisited by genomics.

The genus Serratia has been studied for over a century and includes clinically-important and diverse environmental members. Despite this, there is a paucity of genomic information across the genus and a robust whole genome-based phylogenetic framework is lacking. Here, we have assembled and analysed a representative set of 664 genomes from across the genus, including 215 historic isolates originally used in defining the genus. Phylogenomic analysis of the genus reveals a clearly-defined population structure which displays deep divisions and aligns with ecological niche, as well as striking congruence between historical biochemical phenotyping data and contemporary genomics data. We highlight the genomic, phenotypic and plasmid diversity of Serratia, and provide evidence of different patterns of gene flow across the genus. Our work provides a framework for understanding the emergence of clinical and other lineages of Serratia.

Acoustic component programming in children with cochlear implants using electrocochleography.

OBJECTIVE: Objective measurements improve reliability and effectiveness of hearing assessment and cochlear implant (CI) programming in young children. In CI recipients with acoustic hearing in the implanted ear, electrocochleography (ECochG) can be conducted using intracochlear electrodes. The cochlear microphonic (CM) portion of ECochG has been shown to correlate with pure-tone audiometric thresholds in adult and paediatric CI recipients. Our goal was to determine if ECochG thresholds can be used to appropriately fit the acoustic component to the implanted ear in children. DESIGN: Prospective. STUDY SAMPLE: Eight children (aged 3.5-15.5 years, 10 ears) implanted with Advanced Bionics HiFocus Mid-Scala electrode array were recruited. CI ear acoustic thresholds were measured behaviourally and via ECochG. Two acoustic component enabled CI programs were created using the two sets of thresholds. Age and language appropriate speech outcomes and subjective feedback were obtained. RESULTS: Speech scores were equivalent with the behavioural and ECochG thresholds programs. Subjectively, the ECochG thresholds program was preferred by 7/8 subjects. One subject preferred to use an electric only program. CONCLUSION: Our data suggest that ECochG thresholds can be used to supplement the behavioural clinical methods and aid the reliable fitting of the acoustic component in paediatric CI recipients.

The ecological impact of a bacterial weapon: microbial interactions and the Type VI secretion system.

Bacteria inhabit all known ecological niches and establish interactions with organisms from all kingdoms of life. These interactions are mediated by a wide variety of mechanisms and very often involve the secretion of diverse molecules from the bacterial cells. The Type VI secretion system (T6SS) is a bacterial protein secretion system that uses a bacteriophage-like machinery to secrete a diverse array of effectors, usually translocating them directly into neighbouring cells. These effectors display toxic activity in the recipient cell, making the T6SS an effective weapon during inter-bacterial competition and interactions with eukaryotic cells. Over the last two decades, microbiology research has experienced a shift towards using systems-based approaches to study the interactions between diverse organisms and their communities in an ecological context. Here, we focus on this aspect of the T6SS. We consider how our perspective of the T6SS has developed and examine what is currently known about the impact that bacteria deploying the T6SS can have in diverse environments, including niches associated with plants, insects and mammals. We consider how T6SS-mediated interactions can affect host organisms by shaping their microbiota, as well as the diverse interactions that can be established between different microorganisms through the deployment of this versatile secretion system.

Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases.

Maturation of [NiFe]-hydrogenases often involves specific proteases responsible for cleavage of the catalytic subunits. Escherichia coli HycI is the protease dedicated to maturation of the Hydrogenase-3 isoenzyme, a component of formate hydrogenlyase-1. In this work, it is demonstrated that a Pectobacterium atrosepticum HycI homologue, HyfK, is required for hydrogenase-4 activity, a component of formate hydrogenlyase-2, in that bacterium. The P. atrosepticum ΔhyfK mutant phenotype could be rescued by either P. atrosepticum hyfK or E. coli hycI on a plasmid. Conversely, an E. coli ΔhycI mutant was complemented by either E. coli hycI or P. atrosepticum hyfK in trans. E. coli is a rare example of a bacterium containing both hydrogenase-3 and hydrogenase-4, however the operon encoding hydrogenase-4 has no maturation protease gene. This work suggests HycI should be sufficient for maturation of both E. coli formate hydrogenlyases, however no formate hydrogenlyase-2 activity was detected in any E. coli strains tested here.

Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness.

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram-negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.

Comparison of Pure-Tone Thresholds and Cochlear Microphonics Thresholds in Pediatric Cochlear Implant Patients.

OBJECTIVES: In adult cochlear implant patients, conventional audiometry is used to measure postoperative residual hearing which requires active listening and patient feedback. However, audiological measurements in pediatric cochlear implant patients are both challenging as well as time consuming. Intracochlear electrocochleography (ECOG) offers an objective and a time-efficient method to measure frequency-specific cochlear microphonic or difference thresholds (CM/DIF) thresholds that closely approximate auditory thresholds in adult cochlear implant patients. The correlation between CM/DIF and behavioral thresholds has not been established in pediatric cochlear implant patients. In the present study, CM/DIF thresholds were compared with audiometric thresholds in pediatric cochlear implant patients with postoperative residual hearing. DESIGN: Thirteen (11 unilateral and 2 bilateral) pediatric cochlear implant patients (mean age = 9.2 years ± 5.1) participated in this study. Audiometric thresholds were estimated using conventional, condition play, or visual reinforcement audiometry. A warble tone stimulus was used to measure audiometric thresholds at 125, 250, 500, 1000, and 2000 Hz. ECOG waveforms were elicited using 50-msec acoustic tone-bursts. The most apical intracochlear electrode was used as the recording electrode with an extra-cochlear ground electrode. The ECOG waveforms were analyzed to determine CM/DIF thresholds that were compared with pediatric cochlear implant patients' audiometric thresholds. RESULTS: The results show a significant correlation (r = 0.77, p < 0.01) between audiometric and CM/DIF thresholds over a frequency range of 125 to 2000 Hz in pediatric cochlear implant patients. Frequency-specific comparisons revealed a correlation of 0.82, 0.74, 0.69, 0.41, and 0.32 between the audiometric thresholds and CM/DIF thresholds measured at 125, 250, 500, 1000, and 2000 Hz, respectively. An average difference of 0.4 dB (±14 dB) was measured between the audiometric and CM/DIF thresholds. CONCLUSIONS: Intracochlear ECOG can be used to measure CM/DIF thresholds in pediatric cochlear implant patients with residual hearing in the implanted ear. The CM/DIF thresholds are similar to the audiometric thresholds at lower test frequencies and offer an objective method to monitor residual hearing in difficult-to-test pediatric cochlear implant patients.

A family of Type VI secretion system effector proteins that form ion-selective pores.

Type VI secretion systems (T6SSs) are nanomachines widely used by bacteria to deliver toxic effector proteins directly into neighbouring cells. However, the modes of action of many effectors remain unknown. Here we report that Ssp6, an anti-bacterial effector delivered by a T6SS of the opportunistic pathogen Serratia marcescens, is a toxin that forms ion-selective pores. Ssp6 inhibits bacterial growth by causing depolarisation of the inner membrane in intoxicated cells, together with increased outer membrane permeability. Reconstruction of Ssp6 activity in vitro demonstrates that it forms cation-selective pores. A survey of bacterial genomes reveals that genes encoding Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 and similar proteins represent a new family of T6SS-delivered anti-bacterial effectors.

The plant pathogen Pectobacterium atrosepticum contains a functional formate hydrogenlyase-2 complex.

Pectobacterium atrosepticum SCRI1043 is a phytopathogenic Gram-negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly understood membrane-bound enzyme termed formate hydrogenlyase-2 (FHL-2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL-2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL-2 complex was shown to comprise a rare example of an active [NiFe]-hydrogenase-4 (Hyd-4) isoenzyme, itself linked to an unusual selenium-free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane arm of FHL-2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL-2 function and structure in particular.

A New Front in Microbial Warfare-Delivery of Antifungal Effectors by the Type VI Secretion System.

Microbes typically exist in mixed communities and display complex synergistic and antagonistic interactions. The Type VI secretion system (T6SS) is widespread in Gram-negative bacteria and represents a contractile nano-machine that can fire effector proteins directly into neighbouring cells. The primary role assigned to the T6SS is to function as a potent weapon during inter-bacterial competition, delivering antibacterial effectors into rival bacterial cells. However, it has recently emerged that the T6SS can also be used as a powerful weapon against fungal competitors, and the first fungal-specific T6SS effector proteins, Tfe1 and Tfe2, have been identified. These effectors act via distinct mechanisms against a variety of fungal species to cause cell death. Tfe1 intoxication triggers plasma membrane depolarisation, whilst Tfe2 disrupts nutrient uptake and induces autophagy. Based on the frequent coexistence of bacteria and fungi in microbial communities, we propose that T6SS-dependent antifungal activity is likely to be widespread and elicited by a suite of antifungal effectors. Supporting this hypothesis, homologues of Tfe1 and Tfe2 are found in other bacterial species, and a number of T6SS-elaborating species have been demonstrated to interact with fungi. Thus, we envisage that antifungal T6SS will shape many polymicrobial communities, including the human microbiota and disease-causing infections.

The Type VI secretion system: a versatile bacterial weapon.

The Type VI secretion system (T6SS) is a protein nanomachine that is widespread in Gram-negative bacteria and is used to translocate effector proteins directly into neighbouring cells. It represents a versatile bacterial weapon that can deliver effectors into distinct classes of target cells, playing key roles in inter-bacterial competition and bacterial interactions with eukaryotic cells. This versatility is underpinned by the ability of the T6SS to deliver a vast array of effector proteins, with many distinct activities and modes of interaction with the secretion machinery. Recent work has highlighted the importance and diversity of interactions mediated by T6SSs within polymicrobial communities, and offers new molecular insights into effector delivery and action in target cells.

The type VI secretion system deploys antifungal effectors against microbial competitors.

Interactions between bacterial and fungal cells shape many polymicrobial communities. Bacteria elaborate diverse strategies to interact and compete with other organisms, including the deployment of protein secretion systems. The type VI secretion system (T6SS) delivers toxic effector proteins into host eukaryotic cells and competitor bacterial cells, but, surprisingly, T6SS-delivered effectors targeting fungal cells have not been reported. Here we show that the 'antibacterial' T6SS of Serratia marcescens can act against fungal cells, including pathogenic Candida species, and identify the previously undescribed effector proteins responsible. These antifungal effectors, Tfe1 and Tfe2, have distinct impacts on the target cell, but both can ultimately cause fungal cell death. 'In competition' proteomics analysis revealed that T6SS-mediated delivery of Tfe2 disrupts nutrient uptake and amino acid metabolism in fungal cells, and leads to the induction of autophagy. Intoxication by Tfe1, in contrast, causes a loss of plasma membrane potential. Our findings extend the repertoire of the T6SS and suggest that antifungal T6SSs represent widespread and important determinants of the outcome of bacterial-fungal interactions.

Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system.

The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. 'Defensive' T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are 'offensive', firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of Serratia marcescens to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in S. marcescens. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities.

Dual Role for DsbA in Attacking and Targeted Bacterial Cells during Type VI Secretion System-Mediated Competition.

Incorporation of disulfide bonds into proteins can be critical for function or stability. In bacterial cells, the periplasmic enzyme DsbA is responsible for disulfide incorporation into many extra-cytoplasmic proteins. The type VI secretion system (T6SS) is a widely occurring nanomachine that delivers toxic effector proteins directly into rival bacterial cells, playing a key role in inter-bacterial competition. We report that two redundant DsbA proteins are required for virulence and for proper deployment of the T6SS in the opportunistic pathogen Serratia marcescens, with several T6SS components being subject to the action of DsbA in secreting cells. Importantly, we demonstrate that DsbA also plays a critical role in recipient target cells, being required for the toxicity of certain incoming effector proteins. Thus we reveal that target cell functions can be hijacked by T6SS effectors for effector activation, adding a further level of complexity to the T6SS-mediated inter-bacterial interactions which define varied microbial communities.

Quantitative Determination of Anti-bacterial Activity During Bacterial Co-culture.

Anti-bacterial activity assays are an important tool in the assessment of the ability of one bacterium to kill or inhibit the growth of another, for example during the study of the Type VI secretion system (T6SS) and the anti-bacterial toxins it secretes. The method we describe here can detect the ability of a bacterial strain to kill or inhibit other bacterial cells in a contact-dependent manner when co-cultured on an agar surface. It is particularly useful since it enumerates the recovery of viable target cells and thus enables quantification of the anti-bacterial activity. We provide a detailed description of how to measure the T6SS-dependent anti-bacterial activity of a bacterium such as Serratia marcescens against a competitor prokaryotic organism, Escherichia coli, and also describe possible variations in the method to allow adaptation to other attacker and target organisms.