Lipopolysaccharide-acylating capacity of the gut microbiota and its potential impact on the immunopathogenesis of HIV infection

Storm-Larsen et al.[1] investigated effects of a probiotic treatment on the capacity of gut microbiota to produce hexa-acylated, penta-acylated and tetra-acylated lipopolysaccharide (LPS) in HIV-infected individuals undergoing antiretroviral therapy (ART). They report that the microbiota producing hexa-acylated LPS are outnumbered (by a factor of 25) by those producing penta-acylated LPS, and the hexa:penta ratio correlates with certain markers of systemic inflammation (neopterin and kynurenine:tryptophan ratio; a measure of tryptophan catabolism) [1]. Interestingly, probiotic treatment for 8 weeks causes a reduction in penta-acylating bacteria with a parallel reduction in the plasma LPS levels. Furthermore, no tetra-acylated LPS-producing bacteria were detected. To the best of our knowledge, this is the first study that has investigated the functional capacity of the gut microbiome for acylating LPS in HIV-infected individuals undergoing ART. LPS is a glycolipid inserted in the outer leaflet of the outer membrane of the bacterial cell wall in Gram-negative bacteria [2]. The bacteria can make numerous modifications in their LPS structure in response to environmental cues [2,3]. The remodelling of LPS enables the bacteria to adapt to their microenvironments, evade the host's immune system and prolong their survival. The lipid A moiety mediates the endotoxic and inflammatory activity of LPS. It comprises a backbone of α1-6 linked diglucosamine to which four to seven acyl chains are attached. Four of the chains (R-3-hydroxymyristate) are attached directly to the diglucosamine at the 2, 3, 2′ and 3′C positions and are called primary acyl chains [3]. The tetra-acylated LPS is transported to the outer membrane wherein secondary acyl chains are attached to the hydroxyl groups of the primary chains. The acyltransferases encoded by the gene LpxL and LpxM add fifth and sixth acyl chains via acyloxyacyl linkages at the 2′ and 3′C positions in a sequential manner generating penta- and/or hexa-acylated LPS, respectively. The bacteria lacking these genes make tetra-acylated LPS [4,5]. Another acyltransferase (PagP) in some bacteria may also add seventh acyl chain at the C2 position making a hepta-acylated LPS [6]. Furthermore, certain bacteria can also switch between tetra-acylated and hexa-acylated forms. For example, Yersinia pestis produces hexa-acylated LPS when it infects fleas (at 20°C–25°C) and tetra-acylated LPS when it infects humans (at 37°C) [7,8]. The bacteria do so by switching off the LpxR gene that encodes a deacylase to remove the secondary acyl chains at a higher temperature. The acyl chains play an essential role in the binding of LPS with, and stimulating, MD2/TLR4 complex [9,10]. Typical hexa-acylated LPS from Escherichia coli K12 is a potent stimulator of TLR4. It is 100 times more active than penta-acylated and hepta-acylated LPS. Tetra-acylated LPS completely lacks endotoxic potential and acts as a TLR4 antagonist. A synthetic tetra-acylated lipid A molecule (Eritoran) derived from nonpathogenic Rhodobacter sphaeroides has been approved for treating inflammatory diseases [11]. The differentially acylated LPS species also exert opposite effects on the development of adaptive cellular immunity. For example, hexa-acylated LPS promotes development of TH1, whereas tetra-acylated LPS promotes development of Treg responses [12]. In recent years, LPS has also been shown to bind and activate caspase 11 in mice and their homologs (caspase 4 and 5) in humans. The activated caspases cause pyroptosis, a kind of cell death accompanied by the release of interleukin (IL)-1β and IL-18 [13,14]. Interestingly, hypo-acylated LPS is also less effective in inducing pyroptosis in human cells [15]. The role of gut microbiota in human health is increasingly being realized. HIV is known to decrease diversity of the microbiota and cause significant perturbations in their normal composition in virus-infected individuals. ART is not able to fully restore the diversity and composition of the microbiota in these patients [16]. Furthermore, infection is accompanied by a depletion of TH17 and TH22 in the gut mucosa resulting in increased intestinal permeability and microbial translocation [17]. Increased concentrations of LPS in the circulation of HIV-infected individuals have been well documented [18]. However, little attention has been paid to the nature of the circulating LPS with respect to the acylation of its lipid moiety. The studies conducted so far have been restricted to measuring its levels in the circulation. It is highly likely that the gut microbiota in HIV-infected individuals have remodelled their LPS to survive in the altered gut microenvironment in these patients. Investigating its acylating capacity is clearly important for a better understanding of the inflammatory potential of increased plasma LPS and its role in the immunopathogenesis of the HIV-induced disease. The results from the Storm-Larsen et al.[1] suggest a high ratio of the gut microbiota producing penta:hexa acylated LPS in HIV-infected individuals undergoing ART. However, the important question of how HIV infection and ART affect this ratio remains unknown because no information is available about this ratio in healthy and treatment-naive HIV-infected individuals. Future studies should address this important issue. The predominance of penta-acylated LPS-producing bacteria in the gut microbiota of HIV-infected individuals undergoing ART suggests that plasma LPS in these patients has relatively low endotoxic potential. It may also act as a competitive inhibitor of hexa-acylated LPS and therefore may attenuate inflammation and immune activation. However, hypo-acylated LPS is also known to inhibit ‘LPS tolerance’. This inhibition may result in the abrogation of protective effects of the tolerance for the host [19]. Clearly, further studies are required to understand the role of hexa and penta-acylated LPS in the immunopathogenesis of AIDS. For future studies, metatranscriptomics strategies should be preferred over metagenomics ones and transcripts of the LPS modifying genes should be quantified because bacteria are known to regulate expression of these genes in response to environmental cues. Furthermore, matrix-assisted laser desorption/ionization-time of flight mass spectroscopy should be used to directly quantify differentially acylated forms of plasma LPS. These investigations may lead to a better understanding of the role of differential LPS acylation in the pathogenesis of HIV and may also lead to novel LPS-based therapeutics for reducing inflammation and immune activation in the virus-infected individuals. It is noteworthy that Eritoran (tetra-acylated TLR4 antagonist) protects mice from lethal infections with Influenza virus and Filovirus [11,20]. It may be useful in other viral infections including HIV. Acknowledgements We thank our colleagues and current members of the laboratory who participated in the discussions on the subject. Conflicts of interest There are no conflicts of interest.

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