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A Detailed Introduction to Bordetella (II)

The constitutive lipid A endotoxin that is found in most enterobacteria is a β-1′,6-linked disaccharide of glucosamine, which is phosphorylated at the 1 and 4′ positions and acylated with primary R-3-hydroxymyristate chains at the 2, 3, 2′ and 3′ positions. The structure is further acylated with secondary laurate and myristate chains in acyloxyacyl linkage at the 2′ and 3′ positions respectively. This hexa-acylated lipid A species can be modified by the regulated addition of a palmitate chain in acyloxyacyl linkage at position 2 to produce a hepta-acylated lipid A species. Studies of immune signalling in human cell lines using chemically synthesized hexa-acylated and hepta-acylated lipid A revealed that palmitoylation attenuates the response to lipid A by 10- to 100-fold. One study indicated that palmitoylation converts lipid A into an endotoxin antagonist because prior treatment of human monocytes or macrophages with hepta-acylated lipid A could block signalguing by the hexa-acylated molecule. On its own, hepta-acylated lipid A was inactive as a signaling molecule in these studies. However, efforts to reproduce these results only served to reinforce the earlier conclusion that palmitoylation merely attenuates lipid A signaling. The authors suggest that variations in the levels of expression of TLR4 and/or its associated factor MD-2 might explain these distinctly different observations. Similar conclusions that palmitoylated lipid A is less active were recently obtained using the corresponding natural lipid A species, which were isolated from bacteria that possessed key lipid A modifying enzymes-lipid A palmitoyltransferase (PagP protein). Palmitoylated lipid A normally coexists as a substoichiometric component with other regulated covalent lipid A modifications, which together can directly benefit the bacterium independently of any effects they might have on lipid A signaling.

The pagP gene responsible for lipid A palmitoylation was first identified in a Salmonella mutant that was found to be sensitive to cationic antimicrobial peptides (CAMPs), which are included among the products of the TLR4 signal transduction pathway. Consequently, the modification of lipid A with a palmitate chain appears, remarkably, to both attenuate the production of CAMPs through the TLR4 pathway and protect the bacterial pathogen from the antimicrobial products of that same pathway. However, it is important to recognize that pagP only provides resistance to certain CAMPs, not all of which are products of the innate immune system. CAMPs are generally unstructured in aqueous solution and encounter bacteria by electrostatic interactions with the negatively charged bacterial surface. The increased hydrophobicity at the membrane interface strips hydrogen bonded water molecules from the peptide bonds to drive folding into α-helical or β-sheet conformations. The induced secondary structure reveals the amphipathic nature of CAMPs, which have opposing polar and hydrophobic faces. The amphipathic structure facilitates the insertion of CAMPs into the hydrocarbon domain of the OM before uptake into the periplasmic space. Ultimately, CAMPs target the inner membrane, where they kill bacterial cells primarily by disrupting the membrane potential. However, the OM provides a significant barrier to CAMP uptake, and lipid A palmitoylation presumably increases the hydrophobic and van der Waals interactions in the OM so as to interfere with CAMP translocation across the bilayer. Neutralization of negative charges in lipid A by modification of the phosphates with positively charged l-4-aminoarabinose and/or phosphoethanolamine substituents similarly provides resistance to CAMPs, but in this case the mechanism involves blocking the initial electrostatic interactions. Only modification of the lipid A acylation pattern by palmitoylation is known to be additionally associated with attenuation of lipid A signaling through the TLR4 pathway. Modification of lipid A by 3-O-deacylation similarly attenuates lipid A signaling, but without any other apparent functional consequences. The functional importance of regulated S-2-hydroxylation at the secondary myristate chain is also unclear, but these two lipid A modifications each provide a hydrogen bond-donating hydroxyl group that might help to stabilize lateral interactions between neighbouring LPS molecules in the OM.

The expression of many of the pathogenic components is regulated in response to environmental stimuli by the Bvg two-component regulatory system, which comprises the histidine kinase sensor protein BvgS and the DNA-binding response regulator protein BvgA. The Bvg system and its role in Bordetella biology have been reviewed elsewhere. Briefly, activation of Bvg results in a marked change in the gene expression profile of Bordetella. Many of the proteins that are implicated in Bordetella pathogenesis are expressed only when the Bvg system is stimulated, which indicates that Bordetella adopt this phenotype (known as the Bvg+ phase) during the infection process. In the absence of an (unknown) environmental stimulus, the bacteria adopt a Bvg phase that is thought to resemble the physiological status of bacteria in the environment. A third phenotypic phase, known as Bvg intermediate (Bvgi), has also been identified, but its role in Bordetella biology is unclear. The Bvg system regulates a spectrum of responses that are dependent on the magnitude and duration of the stimulus sensed by BvgS. A recent study used B. pertussis genome sequence information to conduct microarray analysis of the regulation of expression of 181 selected genes by the Bvg system. This limited study identified several new members of the Bvg regulon, so it is likely that other novel genes identified by the genome sequence project also form part of this regulon- a complete definition of which requires a comprehensive study using whole-genome microarrays. The same study also showed that the expression of some genes varied dependent on the modulating signal used to modify Bvg activity. It is therefore likely that Bvg functions with other mechanisms to regulate the expression of some genes. It will be intriguing, for example, to discover how the Bvg regulon is related to the set of genes regulated by other two component systems, such as the ris system.

To be continued in Part III…


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