Experimental acute Clostridium perfringens type D enterotoxemia in sheep is not characterized by specific renal lesions.

Type D enterotoxemia, caused by Clostridium perfringens epsilon toxin (ETX), is one of the most economically important clostridial diseases of sheep. Acute type D enterotoxemia is characterized by well-documented lesions in the nervous, cardiocirculatory, and pulmonary systems. However, discrepancies and confusion exist as to whether renal lesions are part of the spectrum of lesions of this condition, which is controversial considering that for many decades it has been colloquially referred to as "pulpy kidney disease." Here, the authors assess renal changes in an experimental model of acute type D enterotoxemia in sheep and evaluate the possible role of ETX in their genesis. Four groups of 6 sheep each were intraduodenally inoculated with either a wild-type virulent C. perfringens type D strain, an etx knockout mutant unable to produce ETX, the etx mutant strain complemented with the wild-type etx gene that regains the ETX toxin production, or sterile culture medium (control group). All sheep were autopsied less than 24 hours after inoculation; none of them developed gross lesions in the kidneys. Ten predefined histologic renal changes were scored in each sheep. The proportion of sheep with microscopic changes and their severity scores did not differ significantly between groups. Mild intratubular medullary hemorrhage was observed in only 2 of the 12 sheep inoculated with the wild-type or etx-complemented bacterial strains, but not in the 12 sheep of the other 2 groups. The authors conclude that no specific gross or histologic renal lesions are observed in sheep with experimental acute type D enterotoxemia.

An assessment of bacterial contamination of indirect ophthalmoscopes and condensing lenses used in clinical practice: A multi-center study.

PURPOSE: To investigate bacterial contamination of indirect ophthalmoscopes and condensing lenses used in three UK veterinary referral centers, and the impact of an implemented cleaning protocol. METHODS: Bacteriology samples from 10 indirect ophthalmoscopes and 10 condensing lenses were taken at each center (n = 30 T0), before initiating one of three cleaning frequencies (every 2 weeks/once weekly/daily) for 28 days. The most contaminated indirect ophthalmoscope and condensing lens from each center were re-sampled 30 min prior to (T1; n = 9) and 30 min after (T2; n = 9) the final clean. Sensitivity testing was completed using MIC. RESULTS: Seventy-three isolates representing 15 different bacterial populations (genus/species) were cultured from 36 of 48 (75%) swabs tested. The most frequently cultured isolates were Staphylococcus spp. 30%, Micrococcus 22%, and Bacillus 14%. Pseudomonas aeruginosa, Pantoea, and Staphylococcus pseudintermedius demonstrated resistance to >50% of antibiotics against which they were tested. Eighty-three percent of T0 samples (54 isolates across 11 species, median 2 isolates/swab), all T1 samples (15 isolates across 8 species, median 2 isolates/swab), and 22% of T2 samples (4 isolates across 4 species, median 0 isolates/swab) were contaminated. Head contact points were most contaminated irrespective of time point. A T1 sample was 57 times more likely (95% CI: 2.4-1376) to have a positive culture than a T2 sample (p = .01). CONCLUSIONS: Baseline contamination was high, representing a potential source of nosocomial infection in ophthalmic patients and handlers of diagnostic equipment. No center implemented a cleaning protocol prior to this study. Routine cleaning reduces bacterial contamination.

Progressive ulcerative keratitis in dogs in the United Kingdom: Microbial isolates, antimicrobial sensitivity, and resistance patterns.

OBJECTIVES: The objective of the study was to identify bacterial pathogens and their antimicrobial sensitivity profile associated with cases of canine progressive ulcerative keratitis. MATERIALS AND METHODS: Analysis of microbial culture and sensitivity results from dogs with progressive ulcerative keratitis presenting to a UK referral practice between December 2018 and August 2020. RESULTS: Positive bacterial cultures were obtained from 80/148 (54%) of the canine ulcers sampled with 99 bacterial isolates cultured. Streptococcus canis (n = 29), Pseudomonas aeruginosa (n = 19), and Staphylococcus pseudintermedius (n = 16) were the most common isolates. Pseudomonas aeruginosa was more likely to be isolated whether the ulcer was clinically malacic at the time of sampling (OR = 10.1, p < .001). Ulcers treated prior to culture with fusidic acid were 7.6 times more likely to be positive than those treated with any other antimicrobial(s). Bacterial isolates demonstrated resistance against neomycin (85%), fusidic acid (78%), and tetracycline (68%). Conversely, isolates were most likely to be sensitive to gentamicin (88%), ofloxacin (77%), ciprofloxacin (73%), and chloramphenicol (64%). Antimicrobial combinations of chloramphenicol or gentamicin with a fluoroquinolone (ofloxacin or ciprofloxacin) or chloramphenicol combined with gentamicin were the most effective on in vitro analysis (over 90% susceptibility of all isolates). CONCLUSION: The most common bacterial species associated with canine progressive ulcerative keratitis in a UK referral population were S. canis, P. aeruginosa, and S. pseudintermedius. Combination antimicrobial therapy is recommended pending culture and sensitivity results given the varied antimicrobial susceptibility profiles and significant bacterial in vitro resistance to antimicrobial monotherapy.

The ever-expanding tcp conjugation locus of pCW3 from Clostridium perfringens.

The spore-forming, anaerobic Gram positive pathogen Clostridium perfringens encodes many of its disease-causing toxins on closely related conjugative plasmids. Studies of the tetracycline resistance plasmid pCW3 have identified many of the genes involved in conjugative transfer, which are located in the tcp conjugation locus. Upstream of this locus is an uncharacterised region (the cnaC region) that is highly conserved. This study examined the importance in pCW3 conjugation of several highly conserved proteins encoded in the cnaC region. Conjugative mating studies suggested that the SrtD, TcpN and Dam proteins were required for efficient pCW3 transfer between C. perfringens cells from the same strain background. The requirement of these proteins for conjugation was amplified in matings between C. perfringens cells of different strain backgrounds. Additionally, the putative collagen adhesin protein, CnaC, was only required for the optimal transfer of pCW3 between cells of different strain backgrounds. Based on these studies we postulate that CnaC, SrtD, TcpN and Dam are involved in enhancing the transfer frequency of pCW3. These studies have led to a significant expansion of the tcp conjugation locus, which now encompasses a 19 kb region.

Cardiopulmonary Lesions in Sheep Produced by Experimental Acute Clostridium Perfringens Type D Enterotoxemia.

Enterotoxemia caused by Clostridium perfringens type D is one of the most prevalent clostridial diseases of sheep. The lesions of the acute form of this disease, particularly the cerebral lesions, are well characterized; however, detailed descriptions of the cardiac and pulmonary lesions are lacking. Here we describe cardiopulmonary lesions in experimental acute type D enterotoxemia in sheep and determine the role of epsilon toxin (ETX) in the development of these lesions. Four groups of 6 sheep were intraduodenally inoculated with either a wild-type C. perfringens type D strain; its etx knockout mutant, which is unable to produce ETX; the etx mutant complemented with the wild-type etx gene, which regains the ETX toxigenic ability; or sterile culture medium as a control. All sheep were subjected to postmortem examination within 24 hours of inoculation. Lesion scores were compared between groups for pulmonary edema; hydrothorax; ascites; hydropericardium; endocardial, myocardial and epicardial hemorrhages; microscopic lesions of acute myocardial degeneration and necrosis; and myocardial, endocardial, and epicardial edema, hemorrhage, and inflammation. Only sheep inoculated with the wild-type and complemented ETX-toxigenic bacterial strains developed cardiopulmonary lesions, which were present in varying degrees of severity and proportions. These lesions were not present in sheep inoculated with the etx mutant or in the negative control. We conclude that severe acute cardiopulmonary lesions in sheep with experimental enterotoxemia are associated with the capacity of the strains to produce ETX. These changes are likely contributors to the clinical signs and even death of affected animals.

The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in Clostridium perfringens gas gangrene infections.

Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance.

Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.

pCP13, a representative of a new family of conjugative toxin plasmids in Clostridium perfringens.

Conjugative transfer is a major contributor to the dissemination of antibiotic resistance and virulence genes in the human and animal pathogen, Clostridium perfringens. The C. perfringens plasmid pCW3 is the archetype of an extensive family of highly related conjugative toxin and antibiotic resistance plasmids found in this bacterium. These plasmids were thought to constitute the only conjugative plasmid family in C. perfringens. Recently, another series of C. perfringens plasmids, the pCP13-like family, have been shown to harbour important toxin genes, including genes that encode the novel binary clostridial enterotoxin, BEC. Based on early bioinformatics analysis this plasmid family was thought to be non-conjugative. Here we demonstrate that pCP13 is in fact conjugative, transfers at high frequency and that the newly defined Pcp conjugation locus encodes putative homologues of a type 4 secretion system (T4SS), one of which, PcpB4, was shown to be essential for transfer. The T4SS of pCP13 also appears to be evolutionarily related to conjugative toxin plasmids from other clostridia-like species, including Paeniclostridium (formerly Clostridium) sordellii, Clostridioides (formerly Clostridium) difficile and Clostridium botulinum. Therefore, it is clear that there are two distinct families of conjugative plasmids in C. perfringens: the pCW3 family and the pCP13 family. This study has significant implications for our understanding of the movement of toxin genes both within C. perfringens, but also potentially to other pathogenic clostridia.

Antibiotic resistance plasmids and mobile genetic elements of Clostridium perfringens.

Clostridium perfringens is an anaerobic bacterium that is a major human and animal pathogen. The key features of C. perfringens-mediated infections are that disease pathogenesis involves the production of protein toxins and that disease epidemiology generally involves the production of environmentally resistant endospores. Many of the toxins involved in these diseases are encoded on conjugative plasmids that are closely related to the paradigm tetracycline resistance plasmid pCW3. This plasmid encodes the Tet(P) tetracycline resistance determinant, and the tcp locus, which mediates conjugative transfer and is also present on the toxin plasmids. In addition to being directly responsible for the widely dispersed distribution of the Tet(P) determinant, which is not located on a transposable genetic element, this family of conjugative plasmids facilitates the spread of other mobile resistance elements. These elements include the chloramphenicol resistance integrative mobilisable elements typified by Tn4451, the bacitracin resistance integrative conjugative element typified by ICECp1, and the lincomycin resistance transferable insertion sequence typified by tISCpe8. Each of these elements are found on conjugative plasmids that are closely related to pCW3, providing evidence that this large plasmid family has a key role in the distribution of antibiotic resistance genes in C. perfringens.

The incidence of Clostridioides difficile and Clostridium perfringens netF-positive strains in diarrheic dogs.

The aim of this study was to examine the incidence of Clostridioides (previously Clostridium) difficile and Clostridium perfringens in the feces of diarrheic and non-diarrheic dogs. Also, the presence of other common canine enteropathogens was examined. Toxigenic C. difficile and C. perfringens positive for the NetF-encoding gene (netF) were detected in 11 (11.9%) and seven (7.6%) diarrheic dogs, respectively. Three dogs were diagnosed simultaneously with toxigenic C. difficile and netF-positive C. perfringens. Among other enteropathogens, Giardia sp. was the most common agent detected in dogs positive for toxigenic C. difficile or netF-positive C. perfringens. The results suggest that C. difficile and C. perfringens occur more frequently as a primary cause of diarrhea.

Expansion of the Clostridium perfringens toxin-based typing scheme.

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, ß-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not ß-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.

Evidence that compatibility of closely related replicons in Clostridium perfringens depends on linkage to parMRC-like partitioning systems of different subfamilies.

Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRCA-J) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRCC or parMRCD homologues or different combinations of parMRCA, parMRCC and parMRCD family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRCC or parMRCD homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the absence of selection, with the exception of strains housing parMRCC and parMRCD combinations, which showed a minor incompatibility phenotype. In conclusion, we have provided the first direct evidence of plasmid incompatibility in Clostridium spp. and have shown experimentally that the compatibility of conjugative C. perfringens plasmids correlates with the presence of parMRC-like partitioning systems of different phylogenetic subfamilies.

Outcomes and prognostic factors of surgical treatments for brachycephalic obstructive airway syndrome in 3 breeds.

OBJECTIVE: To determine prognostic indicators for the surgical treatment of brachycephalic obstructive airway syndrome (BOAS) and to compare the prognosis of 2 multilevel surgical procedures. STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Client-owned pugs, French bulldogs, and bulldogs (n = 50). METHODS: Noninvasive whole-body barometric plethysmography (WBBP) was used to assess respiratory function before, 1 month and 6 months after upper airway corrective surgery. Postoperatively, BOAS indices (ie, ascending severity score generated from WBBP data, 0%-100%) that equaled to or exceeded the cut-off values of BOAS in the diagnostic models were considered to have a "poor prognosis." A multivariate logistic regression was used to assess predictors for prognosis. RESULTS: The median BOAS indices decreased after surgery (from 76% to 63%, P < .0001), although dogs with indices in this range would still be considered clinically affected. Age (odds ratios [OR] = 0.96, 95% confidence interval [CI]: 0.93-0.99, P < .05), body condition (OR = 0.06, 95% CI: 0.01-0.39, P < .01), laryngeal collapse (OR = 6.1, 95% CI: 1-37.22, P < .05), and surgical techniques (OR = 7.94, 95% CI: 1.17-54.01, P < .05) were associated with postoperative prognosis. The multivariate model suggests modified multilevel surgery (MMS) may have a better outcome than traditional multilevel surgery (TMS) (P = .034). The positive predictive value of the logistic model was 84% (95% CI: 68-94%) and the area under the receiver operating characteristic (ROC) curve was 89% (95% CI: 78-99%, P <.0001). CONCLUSIONS: Younger age, normal body condition, presence of laryngeal collapse, and treatment with TMS were negative prognostic factors after surgical treatment of BOAS. MMS is recommended, particularly in dogs with a higher probability of poor prognosis.

Analysis of the virulence-associated RevSR two-component signal transduction system of Clostridium perfringens.

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes human gas gangrene (clostridial myonecrosis) and food poisoning. Early studies showed that virulence was regulated by the VirSR two-component signal transduction system. However, our identification of the RevR orphan response regulator indicated that more than one system was involved in controlling virulence. To further characterize this virulence-associated regulator, gel mobility shift experiments, coupled with DNase I footprinting, were used to identify the RevR DNA binding sequence. Bioinformatics analysis suggested that an orphan sensor histidine kinase, CPE1757 (renamed RevS), was the cognate sensor of RevR. Interaction between RevS and RevR was demonstrated by use of a bacterial two-hybrid system and validated by protein-protein interaction studies using biolayer interferometry. To assess the involvement of RevS in virulence regulation, the revS gene was inactivated by Targetron insertion. When isogenic wild-type, revS and complemented revS strains were tested in a mouse myonecrosis model, the revS mutant was found to be attenuated in virulence, which was similar to the attenuation observed previously with the revR mutant. However, transcriptional analysis of selected RevR-regulated genes in the revS mutant revealed a different pattern of expression to a revR mutant, suggesting that the RevSR system is more complex than originally thought. Taken together, the results have led to the identification and characterization of the two essential parts of a new regulatory network that is involved in the regulation of virulence in C. perfringens.

Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX.

The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site-specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA-binding motifs. We have solved the solution structure of residues 1-120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX1-120 , we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into (15) N-labeled TnpX1-120 and observed progressive nuclear magnetic resonance chemical shift perturbations using (15) N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data-driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain.

Plasmid partitioning systems of conjugative plasmids from Clostridium perfringens.

Many pathogenic strains of Clostridium perfringens carry several highly similar toxin or antibiotic resistance plasmids that have 35 to 40 kb of very closely related syntenous sequences, including regions that carry the genes encoding conjugative transfer, plasmid replication and plasmid maintenance functions. Key questions are how are these closely related plasmids stably maintained in the same cell and what is the basis for plasmid incompatibility in C. perfringens. Comparative analysis of the Rep proteins encoded by these plasmids suggested that this protein was not the basis for plasmid incompatibility since plasmids carried in a single strain often encoded an almost identical Rep protein. These plasmids all carried a similar, but not identical, parMRC plasmid partitioning locus. Phylogenetic analysis of the deduced ParM proteins revealed that these proteins could be divided into ten separate groups. Importantly, in every strain that carried more than one of these plasmids, the respective ParM proteins were from different phylogenetic groups. Similar observations were made from the analysis of phylogenetic trees of the ParR proteins and the parC loci. These findings provide evidence that the basis for plasmid incompatibility in the conjugative toxin and resistance plasmid family from C. perfringens resides in subtle differences in the parMRC plasmid partitioning loci carried by these plasmids.

Toxin B is essential for virulence of Clostridium difficile.

Clostridium difficile is the leading cause of infectious diarrhoea in hospitals worldwide, because of its virulence, spore-forming ability and persistence. C. difficile-associated diseases are induced by antibiotic treatment or disruption of the normal gastrointestinal flora. Recently, morbidity and mortality resulting from C. difficile-associated diseases have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns. Since 2002, epidemic toxinotype III NAP1/027 strains, which produce high levels of the major virulence factors, toxin A and toxin B, have emerged. These toxins have 63% amino acid sequence similarity and are members of the large clostridial glucosylating toxin family, which are monoglucosyltransferases that are pro-inflammatory, cytotoxic and enterotoxic in the human colon. Inside host cells, both toxins catalyse the transfer of glucose onto the Rho family of GTPases, leading to cell death. However, the role of these toxins in the context of a C. difficile infection is unknown. Here we describe the construction of isogenic tcdA and tcdB (encoding toxin A and B, respectively) mutants of a virulent C. difficile strain and their use in the hamster disease model to show that toxin B is a key virulence determinant. Previous studies showed that purified toxin A alone can induce most of the pathology observed after infection of hamsters with C. difficile and that toxin B is not toxic in animals unless it is co-administered with toxin A, suggesting that the toxins act synergistically. Our work provides evidence that toxin B, not toxin A, is essential for virulence. Furthermore, it is clear that the importance of these toxins in the context of infection cannot be predicted exclusively from studies using purified toxins, reinforcing the importance of using the natural infection process to dissect the role of toxins in disease.

RAB-like 2 has an essential role in male fertility, sperm intra-flagellar transport, and tail assembly.

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.

Utility of the clostridial site-specific recombinase TnpX to clone toxic-product-encoding genes and selectively remove genomic DNA fragments.

TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.