- Volume 153, Issue 6, 2007

The FimA fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. In this study, the four open reading frames (ORF1, ORF2, ORF3 and ORF4) downstream of the fimbrilin gene (fimA) in strain ATCC 33277 were examined. ORF2, ORF3 and ORF4 were demonstrated to encode minor components of the fimbriae and were therefore renamed fimC, fimD and fimE, respectively. Immunoblotting analyses revealed that inactivation of either fimC or fimD by an ermF-ermAM insertion, but not inactivation of ORF1, was accompanied by concomitant loss of the products from the downstream genes, raising the possibility that fimC, fimD and fimE constitute a transcription unit. The fimE mutant produced FimC and FimD, but fimbriae purified from it contained neither protein, suggesting that FimE is required for the assembly of FimC and FimD onto the fimbrilin (FimA) fibre. The fimC, fimD and fimE mutants lost autoaggregation abilities. Fimbriae purified from these three mutants showed attenuated binding activities to glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis and to two extracellular matrix proteins, fibronectin and type I collagen. These results suggest that FimE, as well as FimC and FimD, play critical roles in the adhesive activities of the mature FimA fimbriae in P. gingivalis.

Vibrio vulnificus is a bacterial species that is virulent for humans and fish. Human isolates are classified into biotypes 1 and 3 (BT1 and BT3) and fish isolates into biotype 2 (BT2). However, a few human infections caused by BT2 isolates have been reported worldwide (zoonosis). These BT2 human isolates belong to serovar E (SerE), which is also present in diseased fish. The aim of the present work was to characterize a new BT2 serovar [serovar A (SerA)], which emerged in the European fish-farming industry in 2000, by means of phenotypic, serological and genetic [plasmid profiling, ribotyping and random amplified polymorphic DNA (RAPD)] methodologies. The results confirmed that SerA constitutes a homogeneous O-serogroup within the species that shares plasmidic information with SerE. Like SerE, this new serogroup was resistant to fresh fish serum, as well as being highly virulent for fish. In contrast, it was sensitive to human serum and avirulent for mice, even after pretreatment with iron. The two serovars presented different biochemical profiles as well as specific patterns by ribotyping and RAPD analysis. In conclusion, SerA seems to constitute a different clonal group that has recently emerged within the species V. vulnificus, with pathogenic potential for fish but not for humans.

Chlamydia trachomatis is an obligate intracellular bacterium and acquires both building blocks and energy from host cells for growth. The fatty acid-binding protein (FABP) plays an important role in uptake of long-chain fatty acids (LCFA) and energy metabolism by eukaryotic cells. The roles of FABP and LCFA in chlamydial infection were evaluated. Infection of liver cells with chlamydial organisms promoted fatty acid uptake by the infected cells, suggesting that LCFA may benefit chlamydial growth. Introduction of FABP into the liver cells not only enhanced fatty acid uptake, but also increased chlamydial intravacuolar replication and maturation. The FABP-enhanced chlamydial intracellular growth was dependent on the host cell uptake of fatty acids. These results have demonstrated that C. trachomatis can productively infect liver cells and utilize FABP-transported LCFA for its own biosynthesis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic enteric pathogen of worldwide importance and pigs are a significant reservoir of human infection. Signature-tagged transposon mutagenesis (STM) was used to identify genes required by S. Typhimurium to colonize porcine intestines. A library of 1045 signature-tagged mutants of S. Typhimurium ST4/74 NalR was screened following oral inoculation of pigs in duplicate. A total of 119 attenuating mutations were identified in 95 different genes, many of which encode known or putative secreted or surface-anchored molecules. A large number of attenuating mutations were located within Salmonella pathogenicity islands (SPI)-1 and -2, confirming important roles for type III secretion systems (T3SS)-1 and -2 in intestinal colonization of pigs. Roles for genes encoded in other pathogenicity islands and islets, including the SPI-6-encoded Saf atypical fimbriae, were also identified. Given the role of secreted factors and the protection conferred against other pathogens by vaccination with extracellular and type III secreted proteins, the efficacy of a secreted protein vaccine from wild-type S. Typhimurium following intramuscular vaccination of pigs was evaluated. Serum IgG responses against type III secreted proteins were induced following vaccination and a significant reduction in faecal excretion of S. Typhimurium was observed in the acute phase of infection compared to mock-vaccinated animals. Vaccination with secreted proteins from an isogenic S. Typhimurium prgH mutant produced comparable levels of protection to vaccination with the preparation from the parent strain, indicating that protection was not reliant on T3SS-1 secreted proteins. The data provide valuable information for the control of Salmonella in pigs.

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the ‘translocon’ of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by coils. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of ΔescC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB–LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K s) of a reference strain was about 15 μM in glucose-limited chemostat culture. Disruption of mstA resulted in a two- to fivefold reduction in affinity for glucose and led to expression of a low-affinity glucose transport gene, mstC, at high dilution rate. The effect of mstA disruption was more subtle at low and intermediate dilution rates, pointing to some degree of functional redundancy in the high-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h−1, 0.14 h−1 and 0.20 h−1). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays, and analysed for expression of mstA and two other transporter genes, mstC and mstF. The capacity for glucose uptake (v max) of both strains was significantly reduced at low dilution rate. The glucose uptake assays revealed complex uptake kinetics. This impeded accurate determination of maximum specific uptake rates (v max) and apparent affinity constants () at intermediate and high dilution rate. Two high-affinity glucose transporter genes, mstA and mstF, were expressed at all three dilution rates in chemostat cultures, in contrast to batch culture, where only mstC was expressed. Expression patterns of the three transporter genes suggested differential regulation and functionality of their products.

Ubiquinones (UQs) and menaquinones (MKs) perform distinct functions in Escherichia coli. Whereas, in general, UQs are primarily involved in aerobic respiration, the MKs serve as electron carriers in anaerobic respiration. Both UQs and MKs can accept electrons from various dehydrogenases, and may donate electrons to different oxidases. Hence, they play a role in maintaining metabolic flexibility in E. coli whenever this organism has to adapt to conditions with changing redox characteristics, such as oxygen availability. Here, the authors report on the changes in both the size and the redox state of the quinone pool when the environment changes from being well aerated to one with low oxygen availability. It is shown that such transitions are accompanied by a rapid increase in the demethylmenaquinone pool, and a slow increase in the MK pool. Moreover, in exponentially growing cultures in a well-shaken Erlenmeyer flask, it is observed that the assumption of a pseudo-steady state does not hold with respect to the redox state of the quinone pool.

Modified β-cyclodextrins have been shown previously to enhance sterol conversion to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by growing Mycobacterium spp. The enhancement effect was mainly attributed to steroid solubilization by the formation of inclusion complexes with modified cyclodextrins. In this work, the influence of randomly methylated β-cyclodextrin (MCD) on the growth, AD- and ADD-producing activity, cell wall (CW) composition and ultrastructure of sterol-transforming Mycobacterium sp. VKM Ac-1816D was studied. The specific growth rate of the strain on glycerol increased in the presence of MCD (20–100 mM). Washed cells grown in the presence of MCD (20–40 mM) expressed 1.6-fold higher ADD-producing activity than did the cells grown without MCD, and their adhesiveness differed. Electron microscopy showed MCD-mediated CW exfoliation and accumulation of membrane-like structures outside the cells, while preserving cells intact. The analysis of CW composition revealed both a decrease in the proportion of extractable lipids and a considerable shift in fatty acid profile resulting from MCD action. The MCD-mediated enhancement of mycolic and fatty acids content was observed outside the cells. The total secreted protein level rose 2.4-fold, and the extracellular 3-hydroxysteroid oxidase activity 3.2-fold. The composition of the CW polysaccharide was not altered, while the overall proportion of the carbohydrates in the CW of the MCD-exposed mycobacteria increased. The results showed that the multiple mechanisms of MCD-mediated intensification of sterol to AD(D) conversion by mycobacteria include not only solubilization of steroids, but also the increase of CW permeability for both steroids and soluble nutrients, disorganization of the lipid bilayer and the release of steroid-transforming enzymes weakly associated with the CW.