Ultrastructural Analysis of the Rugose Cell Envelope of a Member of the Pasteurellaceae Family

Abstract

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wildtype and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Inner-membrane protein MorC is involved in fimbriae production and biofilm formation in Aggregatibacter actinomycetemcomitans

“…Disruption of this secretion system results in a reduction of the virulence potential of a number of bacteria including Klebsiella pneumoniae, Proteus mirabilis, Citrobacter rodentium and Salmonella enterica (Burall et al, 2004;Selkrig et al, 2012;Struve et al, 2003). The TamA/B complex is required for the efficient secretion of specific autotransporter proteins to the outer membrane in Enterobacteriaceae (Selkrig et al, 2012), in contrast to what is observed in morC mutants of A. actinomycetemcomitans, which express WT levels of known autotransporters (Smith et al, 2015a), which can easily be visualized by electron microscopy (Azari et al, 2013;Gallant et al, 2008).…”

“…As a member of the family Pasteurellaceae, which includes Haemophilus and Pasteurella spp., A. actinomycetemcomitans displays a rugose outer-membrane morphology in contrast to the flat-membrane morphology displayed by other bacteria (Azari et al, 2013). Loss of morphogenesis protein C (MorC) in the membrane results in conversion of the membrane morphology from rugose to flat, decreasing the surface area of the outer membrane by up to 20 % (Azari et al, 2013;Gallant et al, 2008). In addition to this dramatic morphological change, the absence of MorC results in multiple membrane-related effects including a 30 % decrease in cell size, a 70 % increase in autoaggregation, an 86 % impaired secretion of leukotoxin and a fourfold increase in sensitivity to membranedestabilizing agents (Smith et al, 2015a).…”

“…Bam, which does not fully compensate for the absence of MorC. The indirect model is based on the reduction of both the height of the outer-membrane convolutions and the number of connections between the inner and outer membrane associated with the morC mutant strain (Azari et al, 2013). The fourfold reduction in the number of membrane connections and the twofold decrease in the height of the membrane convolutions lead to a more moderate curvature of the outer membrane and a twofold decrease in the volume/area ratio of the periplasmic space.…”

“…This bacterium is also a member of the HACEK family of pathogens (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), which cause culture-negative infective endocarditis (Das et al, 1997). As a member of the family Pasteurellaceae, which includes Haemophilus and Pasteurella spp., A. actinomycetemcomitans displays a rugose outer-membrane morphology in contrast to the flat-membrane morphology displayed by other bacteria (Azari et al, 2013). Loss of morphogenesis protein C (MorC) in the membrane results in conversion of the membrane morphology from rugose to flat, decreasing the surface area of the outer membrane by up to 20 % (Azari et al, 2013;Gallant et al, 2008).…”

“…A. actinomycetemcomitans cells were visualized by transmission electron microscopy based on the method described by Azari et al (2013). Briefly, appropriate strains were streaked for isolation on solid medium.…”