The Cell Wall of the Pathogenic Bacterium Rhodococcus equi Contains Two Channel-Forming Proteins with Different Properties

Abstract

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA Req (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA Req has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB Req [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.Rhodococcus equi (formerly Corynebacterium equi) is recognized as a significant cause of disease in foals between the ages of 1 and 6 months and is responsible for ca. 3% of global foal mortality (13,33). This organism was first reported as a cause of human disease in 1967 (14), since which time it has emerged as an opportunistic pathogen, notably (but not exclusively) of patients with compromised immunity (25,27,33,52). Moreover, it is likely that the incidence of human infection by R. equi has been underestimated through either misdiagnosis or unwarranted dismissal of the organism as a contaminant diphtheroid (52).In both foals and humans, R. equi disease is typically characterized by bronchopneumonia, although extrapulmonary manifestations are also found (13, 33, 52). R. equi is an intracellular pathogen of alveolar macrophages, and the bacterium is known to enter macrophages via complement receptors following deposition of complement component C3 (25). Once within the macrophage, the bacteria resist the host's killing mechanisms and multiply. However, there is currently little information concerning the specific bacterial factors which facilitate entry into or subsequent survival within macrophages. Persistence within macrophages most likely contributes to the in vivo ...

Reconstitution Experiments and Gene Deletions Reveal the Existence of Two-Component Major Cell Wall Channels in the Genus Corynebacterium

“…In line with the situation in Gram-negative bacteria, in members of the CMN group (the mycolata), facilitation of the uptake of nutrients and other chemical substances is presumably also provided by specialized water-filled channels. Channel-forming proteins in the cell wall of mycobacteria (35,36,61), corynebacteria (10,21,30), nocardiae (47), and closely related genera (45,46) support the assumption.…”

PorH, a new channel-forming protein present in the cell wall of Corynebacterium efficiens and Corynebacterium callunae

“…A search for another cell-wall channel revealed the existence of the anion-selective PorB C.glut channel (Costa-Riu et al, 2003a, b). This result indicates that the cell wall of C. glutamicum contains several types of channels, as is the case in the outer membrane of Gram-negative bacteria (Benz, 2001) and in the Gram-positive Rhodococcus equi, which is also a member of the mycolata (Riess et al, 2003). In this study, we inspected the cell walls of C. efficiens and C. callunae, which are closely related to C. glutamicum, for the presence of cell-wall channels using the lipid bilayer technique.…”

Uptake pathways of anionic and cationic photosensitizers into bacteria

“…These cationic entities were shown to enter the bacterial cytosol via the ‘self promoted uptake pathway’ 10,15. In addition to self promoted pathway, ‘protein transport machineries’ present in the bacterial cell envelope could also mediate PS uptake 16. The ‘porin’ class of protein transporters facilitate the uptake of low molecular weight (600–700 Da) hydrophilic compounds 8.…”

“…The ‘porin’ class of protein transporters facilitate the uptake of low molecular weight (600–700 Da) hydrophilic compounds 8. Although the presence of porin proteins was initially reported in the outer membrane of Gram-negative bacteria, ion selective porin proteins have also been identified on Gram-positive bacterial cell walls 16,17. Taken together, it is understood that the functioning of self-promoted uptake pathways and protein transporters are modulated by charged entities such as cations.…”