The Pseudomonas aeruginosa inner membrane protein FimV is among several proteins of unknown function required for type IV pilus-mediated twitching motility, arising from extension and retraction of pili from their site of assembly in the inner membrane. The pili transit the periplasm and peptidoglycan (PG) layer, ultimately exiting the cell through the PilQ secretin. Although fimV mutants are nonmotile, they are susceptible to killing by pilus-specific bacteriophage, a hallmark of retractable surface pili. Here we show that levels of recoverable surface pili were markedly decreased in fimV pilT retraction-deficient mutants compared with levels in the pilT control, demonstrating that FimV acts at the level of pilus assembly. Levels of inner membrane assembly subcomplex proteins PilM/N/O/P were decreased in fimV mutants, but supplementation of these components in trans did not restore pilus assembly or motility. Loss of FimV dramatically reduced the levels of the PilQ secretin multimer through which pili exit the cell, in part due to decreased levels of PilQ monomers, while PilF pilotin levels were unchanged. Expression of pilQ in trans in the wild type or fimV mutants increased total PilQ monomer levels but did not alter secretin multimer levels or motility. PG pulldown assays showed that the N terminus of FimV bound PG in a LysM motif-dependent manner, and a mutant with an in-frame chromosomal deletion of the LysM motif had reduced motility, secretin levels, and surface piliation. Together, our data show that FimV's role in pilus assembly is to promote secretin formation and that this function depends upon its PG-binding domain.Type IV pili (T4P) are thin, long, flexible, and retractable protein filaments. The broad distribution of T4P among bacterial genera correlates with their ability to mediate a wide range of functions, including attachment to surfaces, DNA uptake, and twitching motility. During twitching motility, pili extend from the cell and attach to a surface, and pilus retraction occurs, moving the bacteria forward toward the point of adhesion (41,51). T4P have been classified into two distinct subtypes, T4aP and T4bP, based on differences in structure of the major pilin subunit and in architecture of the assembly systems (3,16,43). T4aP have been characterized extensively in species such as Pseudomonas aeruginosa, Neisseria spp., and Myxococcus xanthus (10,22,23,39,40,46,49,52). T4bP are found predominately in enteric pathogens, where they promote adhesion (37,58) and bacterial aggregation (6).In P. aeruginosa, a large number of genes involved in the regulation, assembly, and dynamics of T4aP function have been identified. These include the major pilin subunit PilA, as well as a conserved set of minor pilin-like proteins (21); the N-methyltransferase/peptidase PilD, essential for processing prepilins into their mature form (53); an inner membrane assembly subcomplex composed of PilM/N/O/P (4, 47); an outer membrane PilQ secretin pore oligomerized with the assistance of the lipoprotein PilF (7, 29); a...

The clinical sensitivity of nucleic acid amplification tests may be determined by analytical sensitivity and inhibitors in patient samples. We established endpoints for detection of propagated Chlamydia trachomatis L2 434, diluted according to swab and urine protocols for APTIMA Combo 2 (AC2), ProbeTec ET (PT), and Amplicor (AMP) assays. AC2 was 1,000-fold more sensitive than PT and 10-fold more sensitive than AMP on mock swab specimens. For urine, AC2 analytical sensitivity was 100-fold greater than those of the other assays. Spiking an aliquot of each clinical-trial sample from 298 women demonstrated inhibition rates in first-void urine (FVU), cervical swabs (CS), and vaginal swabs (VS) of 12.1%, 12.8%, and 10.4% for AMP; 27.2%, 2%, and 2%, for PT; and 0.3%, 1.7%, and 1.3% for AC2. Inhibition of our C. trachomatis spike and the PT or AMP amplification controls from the manufacturers showed less than 50% correlation. Using an infected-patient reference standard (a specimen positive in at least two tests or a single test positive in two of three samples) in AC2, the VS identified 68/69 (98.6%) infected women compared to CS (89.9%) or FVU (81.2%). Significantly fewer women were identified by PT (65.2%, 63.8%, and 66.7%) or AMP (65.2%, 59.4%, and 56.5%) with the three specimens. By individual specimen type, AC2 confirmed virtually all PT-and AMP-positive specimens, but rates of AC2 confirmation by AMP or PT ranged from 62.9 to 80.3%. The AC2 test identified significantly more women infected with C. trachomatis (P ‫؍‬ 0.001). Vaginal swabs appear to be the specimen of choice for screening.Chlamydia trachomatis is one of the most common sexually transmitted infections in the United States and worldwide (17), largely due to high rates of asymptomatic infection in the lower genital tracts of women and men (24). Early diagnosis followed by treatment of this infection can prevent upper genital tract infection, such as pelvic inflammatory disease (21).For the past 10 years, a great deal of research has led to the development and evaluation of sensitive and specific diagnostic tests for C. trachomatis. These newer nucleic acid amplification tests (NAATs) have been commercialized and are now in routine use in many parts of the world. The important aspect of these assays is their capacity to be used on less invasive specimens, such as vaginal swabs (VS) and first-void urine (FVU), which can be self-collected. Studies have shown that NAATs performed on these less invasive samples are able to detect as many or more infected patients than traditional swabs from the urethra or cervix (2-4, 6-10, 19-22, 25-27, 31).The analytical sensitivity of each test and its susceptibility to inhibitors in different specimen types may determine the clinical sensitivity of each test. We compared detection thresholds and determined the inhibitor and infection rates in three different specimens from 298 women by using three commercial assays for C. trachomatis. MATERIALS AND METHODSSpecimens. Three cervical swabs (CS), three VS, and the first 3...

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Collection of meatal swabs could serve as an alternative to urethral swabbing and FCU for the detection of CT and NG.