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.

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.

Lanthanide elements have been recently recognized as “new life metals” yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate . This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog , an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7 . Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.

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)P], 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 -palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P 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 -palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers-palmitoylation and activation of PI4KIIβ, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines.

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 LaIII and EuIII 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 PrIII, or to monitor the depletion of LaIII 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.

23Lanthanides are elements that have been recently recognized as "new life metals" in bacterial 24 metabolism, yet much remains unknown regarding lanthanide acquisition, regulation, and use. 25The lanthanide-dependent methanol dehydrogenase XoxF produces formaldehyde, which is 26 lethal to Methylorubrum extorquens AM1 if allowed to accumulate. This property enabled a 27 transposon mutagenesis study to expand knowledge of the lanthanide-dependent metabolic 28 network. Mutant strains were reconstructed and growth studies were conducted for over 40 29 strains detailing the involvement of 8 novel genes in lanthanide-dependent and independent 30 methanol growth, including a fused ABC-transporter, aminopeptidase, LysR-type transcriptional 31 regulator, putative homospermidine synthase, mxaD homolog (xoxD), porin family protein, and 32 genes of unknown function previously published as orf6 and orf7. Using genetic and biochemical 33 analyses, strains lacking individual genes in the lanthanide transport cluster were characterized 34 and named lut for lanthanide utilization and transport (META1_1778 to META1_1787). 35Consistent with previous reports, we corroborated that a TonB-ABC transport system is required 36 for lanthanide transport to the cytoplasm. However, an additional outer membrane transport 37 mechanism became apparent after longer growth incubations. Additionally, suppressor mutations 38 that rescued growth of the ABC-transporter mutants were identified. Transcriptional reporter 39 fusions were used to show that like iron transport, expression from the TonB-dependent receptor 40 promoter, lutH, is repressed when lanthanides are in excess. Energy dispersive X-ray 41 spectroscopy analysis was used to visualize the localization of lanthanum in wild-type and 42TonB-ABC transport mutant strains and showed for the first time, that M. extorquens AM1 43 stores cytoplasmic lanthanides in mineral form. 44 45 IMPORTANCE 46Understanding the role of lanthanides in bacterial systems is an emergent field of study. Results 47 from this work define mechanisms by which the methylotrophic bacterium, M. extorquens AM1, 48 acquires and accumulates lanthanides. Lanthanides are critical metals for modern technologies 49 and medical applications, yet lanthanide mining is cost-prohibitive and harmful to the 50 environment. Methylotrophs are an attractive platform for the recovery of lanthanides from 51 discarded electronics, mining leachate, and other waste streams as these microorganisms have 52 been proven to sense and transport these metals for use in alcohol oxidation. Further, 53 methylotrophs are effective biotechnological platforms for chemical productions and can use 54 pollutants such as methane, and inexpensive feedstocks such as methanol. Defining the 55 lanthanide acquisition, transport, and accumulation machinery is a step forward in designing a 56 sustainable, clean platform to recover lanthanides in an efficient and less environmentally 57 destructive manner. 58 59Lanthanide metals (Ln) impact the metabolism of many Gra...

Thioloxidoreductase HP0231 of Helicobacter pylori plays essential roles in gastric colonization and related gastric pathology. Comparative proteomics and analysis of complexes between HP0231 and its protein substrates suggested that several Hop proteins are its targets. HP0231 is a dimeric oxidoreductase that functions in an oxidizing Dsb (disulfide bonds) pathway of H. pylori. H. pylori HopQ possesses six cysteine residues, which generate three consecutive disulfide bridges. Comparison of the redox state of HopQ in wild-type cells to that in hp0231-mutated cells clearly indicated that HopQ is a substrate of HP0231. HopQ binds CEACAM1, 3, 5 and 6 (carcinoembryonic antigen-related cell adhesion molecules). This interaction enables T4SS-mediated translocation of CagA into host cells and induces host signaling. Site directed mutagenesis of HopQ (changing cysteine residues into serine) and analysis of the functioning of HopQ variants showed that HP0231 influences the delivery of CagA into host cells, in part through its impact on HopQ redox state. Introduction of a C382S mutation into HopQ significantly affects its reaction with CEACAM receptors, which disturbs T4SS functioning and CagA delivery. An additional effect of HP0231 on other adhesins and their redox state, resulting in their functional impairment, cannot be excluded.

This study analyzed the occurrence of lanthanide-dependent (XoxF type) methanol dehydrogenases in the bacterial community dominated by Proteobacteria inhabiting shale rock. In total, 22 sequence matches of XoxF were identified in the metaproteome of the community. This enzyme was produced by bacteria represented by orders such as Rhizobiales, Rhodobacterales, Rhodospiralles, Burkholderiales and Nitrosomonadales. Among the identified XoxF proteins, seven belonged to XoxF1 clade and 15 to XoxF5 clade. This study is the first to show the occurrence of XoxF proteins in the metaproteome of environmental lithobiontic bacterial community colonizing an underground rock rich in light lanthanides. The presented results broaden our understanding of the ecology of XoxF producing bacteria as well as the distribution and diversity of these enzymes in the natural environment.