Control of type III secretion activity and substrate specificity by the cytoplasmic regulator PcrG

Pathogenic Gram-negative bacteria use syringe-like type III secretion systems (T3SS) to inject effector proteins directly into targeted host cells. Effector secretion is triggered by host cell contact, and before contact is prevented by a set of conserved regulators. How these regulators interface with the T3SS apparatus to control secretion is unclear. We present evidence that the proton motive force (pmf) drives T3SS secretion in Pseudomonas aeruginosa, and that the cytoplasmic regulator PcrG interacts with distinct components of the T3SS apparatus to control two important aspects of effector secretion: (i) It coassembles with a second regulator (Pcr1) on the inner membrane T3SS component PcrD to prevent effectors from accessing the T3SS, and (ii) In conjunction with PscO, it controls protein secretion activity by modulating the ability of T3SS to convert pmf.Many Gram-negative bacterial pathogens rely on a type III secretion system (T3SS) to promote disease by directly injecting effector proteins into the cytoplasm of host cells. This apparatus consists of a base that spans the bacterial envelope and a needle that projects from the base and ends in a specialized tip structure. The bacterium secretes two translocator proteins via the T3SS, which insert into the host cell membrane to form a pore, through which effector proteins are then transferred (1, 2).One of the hallmarks of type III secretion is that export of effector proteins is triggered by host cell contact (3–5). The secretion apparatus is fully assembled before cell contact, but effector secretion is prevented through the concerted action of needle tip-associated proteins and regulators that control secretion from the bacterial cytoplasm.In most systems, the needle tip protein prevents premature effector secretion, most likely by allosterically constraining the T3SS in an effector secretion “off” conformation (6–10). PcrG, the needle tip protein chaperone, as well as PopN, a member of the YopN/MxiC family of proteins, control effector secretion from the bacterial cytoplasm in Pseudomonas aeruginosa. PcrG’s regulatory function is independent of its function in promoting the export of needle tip protein PcrV. Deletion of pcrG or pcrV results in partial deregulation of effector secretion, whereas removal of both genes results in high-level secretion of effectors (8). In some bacteria, the needle tip protein promotes its own export with the aid of a self-chaperoning domain, rather than with a separate export chaperone (11). Recent evidence suggests that in these systems, the needle tip protein itself also regulates effector secretion from the cytoplasm, in addition to its regulatory role at the T3SS needle tip (12). The mechanism of this regulation is unclear.YopN/MxiC family proteins, PopN in P. aeruginosa, are T3SS regulators that are exported once effector secretion is triggered (13–17). These proteins control effector secretion from the bacterial cytoplasm (18–20). P. aeruginosa PopN and the closely related YopN associate with three other proteins that are required to prevent premature effector secretion (21–23). For PopN, these three proteins are Pcr1, Pcr2, and PscB. Pcr2 and PscB form a heterodimeric export chaperone, and Pcr1 is thought to tether the PopN complex to the apparatus (23). The prevailing model for explaining how PopN and related regulators control effector secretion is that they partially insert and plug the secretion channel while being tethered to the T3SS, either directly via a C-terminal interaction or indirectly via a C-terminal–associated protein, i.e., Pcr1 in P. aeruginosa (19, 20). The apparatus component with which these regulators interact is unknown, however.Triggering of effector secretion results in the rapid injection of effector proteins into the host cell (4, 5). How this rapid burst of secretion is energized is a matter of some controversy. The flagellum, which also uses a type III secretion mechanism, uses the proton motive force (pmf) to catalyze the rapid export of flagellar subunits. In fact, secretion is possible in mutants lacking the flagellum-associated ATPase, FliI, if the associated regulatory protein, FliH, is eliminated as well (24–26). The pmf’s contribution to the rate of secretion relative to the ATPase has been questioned in the case of virulence-associated T3SS (27), where removal of the ATPase results in a complete block of secretion (28, 29) that is not alleviated by deletion of the associated FliH homolog (30).Here we present evidence that export via the P. aeruginosa T3SS is energized primarily by the pmf, thereby offering a unified model for how protein secretion is energized in all T3SSs. The cytoplasmic T3SS regulator PcrG controls both the access of effectors to the T3SS and, surprisingly, the secretion activity of the apparatus. These two functions are controlled by separate regions of PcrG. Control of secretion activity involves the central portion of PcrG as well as PscO, which regulate the pmf-dependent export of secretion substrates. Mutants that up-regulate translocator secretion without turning on effector export confirm that effector secretion is not blocked by physical obstruction of the secretion channel. Instead, access of effectors to the T3SS is controlled by the C terminus of PcrG in conjunction with the PopN complex through an interaction with the inner membrane T3SS component PcrD. This protein complex likely blocks an acceptor site for effectors. Thus, PcrG is a multifaceted protein that, along with its export chaperone function, serves as a brake and a switch to control effector secretion.     

Nonadherence to treatment causing acute hospitalizations in people with epilepsy: An observational,prospective study

The aim was to assess the clinical relevance of antiepileptic drug (AED) nonadherence by means of therapeutic drug concentration monitoring (TDM). Two hundred eighty‐two consecutive patients with epilepsy acutely admitted to hospital for seizures were included. Nonadherence was defined as having a serum concentration/dose ratio at admission of <75% of the patient's own control value (probable nonadherence: 50–75%; definite: <50%). Nonadherence was identified in 39% of patients (definite 24%; probable 15%). It was significantly more common in patients with generalized seizures compared to those with focal onset seizures, and in patients <30 years compared to older patients. When specifically asked, 44% of nonadherent patients claimed regular intake. Nonadherence is a major cause of seizure breakthrough in patients with epilepsy, particularly in young adults. Many patients seem to be unaware of missed drug intake. Prompt measurements of AED serum concentrations should be available as part of the emergency care for patients acutely hospitalized for seizures to permit this issue to be thoroughly addressed prior to discharge.     

Postprandial coagulation activation in overweight individuals after weight loss: Acute and long-term effects of a high-monounsaturated fat diet and a low-fat diet

Diet is important in the prevention of cardiovascular disease, and it has been suggested that a high-MUFA diet is more cardioprotective than a low-fat diet. We hypothesised that the postprandial thrombotic risk profile is improved most favourably by a high-MUFA diet compared with a low-fat diet. This was tested in a parallel intervention trial on overweight individuals (aged 28.4 (SD 4.7) years) randomly assigned to a MUFA-diet (35-45% of energy as fat; > 20% as MUFA, n = 21) or a low-fat (LF) diet (20-30% of energy as fat, n = 22) for 6 months after a weight loss of ~ 10%. All foods were provided free of charge from a purpose-built supermarket. Meal tests designed after the same principles were performed before and after the dietary intervention, and blood samples were collected at 8.00 h (fasting), 12.00 h, and 18.00 h and analysed for factor VII coagulant activity (FVII:C), activated FVII, fibrinogen, prothrombin fragment 1 + 2 (F1 + 2), D-dimer, plasminogen activator inhibitor (PAI:Ag), and thrombin activatable fibrinolysis inhibitor. There were significant postprandial increases in F1 + 2 and D-dimer before and after dietary intervention, with significantly lower values after 6 months. No significant differences were observed between the postprandial changes induced by the two diets. The postprandial decrease in FVII:C and PAI:Ag did not differ before and after intervention, irrespective of the diets. Our findings suggest postprandial coagulation activation in overweight subjects with more pronounced acute than long-term effects. We observed similar effects of the MUFA diet and the LF diet on the postprandial prothrombotic risk profile.     

Pre-vaccination incidence of genital warts in 15-23-year-old women and men attending youth clinics in Stockholm, Sweden

Abstract Background: Human papillomavirus (HPV) vaccines were introduced to the market in 2006 and 2007. The present pilot study was designed to examine the incidence of genital warts in the population up to 23 y of age in the county of Stockholm before the start of mass HPV vaccination. Methods: Data from the electronic health records of 9 youth clinics in the county of Stockholm were collected retrospectively for the y 2006-2008. Results: In total, 49,985 patients visited the study youth clinics during 2006-2008. Of these, 1817 were denoted genital warts patients. An extrapolation of the study data was done in an attempt to estimate the annual number of genital warts cases in the full Stockholm County population aged 15-23 y. Results showed that there were approximately 1792 genital warts patients in the age group 15-23 y each year in Stockholm County. Female cases represented approximately 62% of all cases in the age group 15-23 y. The peak incidence was at around 20 y of age for females, while males had a more flattened peak incidence around 19-23 y of age. Conclusion: This pilot study demonstrates that, compared to other reported data, genital warts are at least as common in Sweden as in other countries among 15-23 y old females and males.     

Early release of interalveolar synechiae under general anesthesia through fiberscopic nasal intubation

This article presents a treatment strategy for early release of interalveolar synechiae, aiming to facilitate early oral feeding and prevent temporomandibular joint ankylosis.The treatment results of 2 patients with van der Woude syndrome were retrospectively studied. Both patients underwent early surgical release of interalveolar synechiae under general anesthesia through fiberscopic nasal intubation. The 2 patients were treated at the ages of 6 and 14 days, respectively. The interincisival distances increased from 5 and 6 mm preoperatively to 11 and 10 mm immediately after surgery. This was increased further to 25 and 20 mm at long-term follow-up (6 and 24 months).In conclusion, synechiae between the upper and lower jaws can be safely treated at a very early age under general anesthesia with fiberscopic nasotracheal intubation. The purpose of early intervention in these cases is to facilitate oral feeding and prevent temporomandibular joint ankylosis.