One-Step ARMS-PCR for the Detection of SNPs-Using the Example of the PADI4 Gene.

In eukaryotes, cellular functions are tightly controlled by diverse post-translational modifications (PTMs) of proteins. One such PTM affecting many proteins is the deimination of arginine to citrulline. This process, called citrullination is catalyzed by a group of hydrolases called protein arginine deiminases (PADs), of which five isoforms have been identified. Hypercitrullination, as a result of increased PAD expression or activity, is associated with autoimmune diseases e.g., rheumatoid arthritis, lupus, Alzheimer's disease, ulcerative colitis, multiple sclerosis, and certain cancers. Three common single nucleotide polymorphisms (SNPs) in the PADI4 gene have been described, namely rs874881, rs11203366, and rs11203367, which are thought to affect PAD4 expression and activity. We here compared the suitability of four methods for the screening of SNPs in the PADI4 gene: (i) SYBR-green based real-time polymerase chain reaction followed by high resolution melting curve analysis (HRM-PCR); (ii) PCR followed by detection of restriction fragment length polymorphisms (PCR-RFLP); (iii) conventional tetra-primer amplification refractory mutation system PCR (ARMS-PCR); and (iv) real-time PCR based on the one-step ARMS-PCR. Of these, ARMS-PCR proved to be the most suitable method regarding handling, duration, and cost of experiments. Using the method with SYBR-green based real-time PCR reagents further diminished handling steps and thus potential sources of error.

Biogenesis and function of the autotransporter adhesins YadA, intimin and invasin.

Bacteria often express numerous virulence factors. These virulence factors make them successful pathogens, by e.g. mediating attachment to host cells and thereby facilitating persistence or invasion, or by contributing to the evasion of the host immune system to allow proliferation and spread within the host and in the environment. The site of first contact of Gram negative bacteria with the host is the bacterial outer membrane (OM). Consisting of an asymmetrical lipid bilayer with phospholipids forming the inner, and lipopolysaccharides forming the outer leaflet, the OM harbors numerous integral membrane proteins that are almost exclusively ß-barrel proteins. One distinct family of OM ß-barrel proteins strongly linked to bacterial virulence are the autotransporter (AT) proteins. During the last years huge progress has been made to better understand the mechanisms underlying the insertion of AT proteins into the OM and also AT function for interaction with the host. This review shortly summarizes our current knowledge about outer membrane protein (OMP) and more specifically AT biogenesis and function. We focused on the AT proteins that we haved studied in most detail: i.e. the Yersinia adhesin A (YadA) and invasin of Yersinia enterocolitica (Ye) as well as its homolog intimin (Int) expressed by enteropathogenic Escherichia coli. In addition, this review provides a short outlook about how we could possibly use this knowledge to fight infection.

Deprivation of the Periplasmic Chaperone SurA Reduces Virulence and Restores Antibiotic Susceptibility of Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the ß-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC, of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics.