S-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. S-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. In this review, we focus on S-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. We discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. We highlight recent data on protein S-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. The role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed.

Campylobacter infection in humans is accompanied by severe inflammation of the intestinal mucosa, in contrast to colonization of chicken. The basis for the differential host response is unknown. Toll-like receptors (TLRs) sense and respond to microbes in the body and participate in the induction of an inflammatory response. Thus far, the interaction of Campylobacter with chicken TLRs has not been studied. Here, we investigated the potential of four Campylobacter strains to activate human TLR1/2/6, TLR4, TLR5, and TLR9 and chicken TLR2t2/16, TLR4, TLR5, and TLR21. Live bacteria showed no or very limited potential to activate TLR2, TLR4, and TLR5 of both the human and chicken species, with minor but significant differences between Campylobacter strains. In contrast, lysed bacteria induced strong NF-κB activation through human TLR1/2/6 and TLR4 and chicken TLR2t2/16 and TLR4 but not via TLR5 of either species. Interestingly, C. jejuni induced TLR4-mediated beta interferon in human but not chicken cells. Furthermore, isolated chromosomal Campylobacter DNA was unable to activate human TLR9 in our system, whereas chicken TLR21 was activated by DNA from all of the campylobacters tested. Our data are the first comparison of TLR-induced immune responses in humans and chickens. The results suggest that differences in bacterial cell wall integrity and in TLR responses to Campylobacter LOS and/or DNA may contribute to the distinct clinical manifestation between the species.

Helicobacter pylori, a member of Epsilonproteobacteria, is a Gram-negative microaerophilic bacterium that colonizes gastric mucosa of about 50% of the human population. Although most infections caused by H. pylori are asymptomatic, the microorganism is strongly associated with serious diseases of the upper gastrointestinal tract such as chronic gastritis, peptic ulcer, duodenal ulcer, and gastric cancer, and it is classified as a group I carcinogen. The prevalence of H. pylori infections varies worldwide. The H. pylori genotype, host gene polymorphisms, and environmental factors determine the type of induced disease. Currently, the most common therapy to treat H. pylori is the first line clarithromycin–based triple therapy or a quadruple therapy replacing clarithromycin with new antibiotics. Despite the enormous recent effort to introduce new therapeutic regimens to combat this pathogen, treatment for H. pylori still fails in more than 20% of patients, mainly due to the increased prevalence of antibiotic resistant strains. In this review we present recent progress aimed at designing new anti-H. pylori strategies to combat this pathogen. Some novel therapeutic regimens will potentially be used as an extra constituent of antibiotic therapy, and others may replace current antibiotic treatments. Key points: • Attempts to improve eradication rate of H. pylori infection. • Searching for new drug targets in anti-Helicobacter therapies.

Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low μM (0.1 to 10 μM) range. We designed this assay to follow La ^III and Eu ^III depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as Pr ^III , or to monitor the depletion of La ^III from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS). IMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that induces strong proinflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding, CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], a lipid controlling subsequent proinflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins that were up-regulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were up-regulated, 186 downregulated, and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P₂ precursor. Silencing of PI4KIIβ and PI4KIIα inhibited LPS-induced expression and production of proinflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KIIβ or PI4KIIα; this was dependent on palmitoylation of the kinases. However, the S-palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers S-palmitoylation and activation of PI4KIIβ, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines.