The deletion of efp or its orthologues leads to several different phenotypes. Having demonstrated that EF-P alleviates translational stalling at polyproline-stretches in E. coli, it is now possible to address some of the proteins, which could be responsible for the resulting effects in efp, yjeK and yjeA mutants.
Peng et al.. determined the phenotype of a deletion mutant (∆chvH) in Agrobacterium tumefaciens (Peng et al., 2001). chvH encodes for a homologue to E. coliefp and clusters with
poxA (yjeA) and kamA (homologue to yjeK) in the genome. The chvH deletion leads to avirulence and other pleiotropic effects, which can be complemented by E. coli EF-P (Peng et al., 2001). A. tumefaciens is very particular as it is able to transfer bacterial DNA into the plant host, followed by integration of the DNA into the plant genome and formation of crown galls (Bevan & Chilton, 1982). The tumor-inducing plasmid Ti harbors the VirA/VirG two- component system, which is responsible for the expression of additional vir genes (Leroux et al., 1987; Ronson et al., 1987; Winans et al., 1986). Comparable to CadC, VirG and VirA are only expressed to significant levels, when cells respond to environmental stress conditions like low pH (Peng et al., 2001; Winans et al., 1988). Interestingly, protein amounts of VirA, VirG and other tested Vir proteins are reduced in the chvH mutant (Peng et al., 2001). VirA contains the amino acid sequence PPG, which was previously shown to cause ribosomal stalling (Tanner et al, 2009; Doerfel et al, 2013; this study). Therefore it can be speculated, whether the reduced protein level of the hisidine kinase VirA leads to the observed impaired production of the Vir proteins in the chvH mutant. In addition, also VirB9 (PPQP), VirB10 (PPS, PPA, PPP, PPT), VirB11 (PPT, PPQ), VirE2 (PPP, PPI) and VirD2 (PPD, PSPP) contain a cluster of at least two prolines, indicating that also these amino acid sequences could contribute to ribosomal stalling and to decreased protein levels in the chvH mutant. Furthermore, the deletion of chvH reveals an increased production of a so far uncharacterized protein (Peng et al., 2001). As the transcriptional regulator VirB, which is phosphorylated by VirA, shows a highly reduced protein amount in the chvH mutant (Peng et al, 2001), the unknown protein perhaps acts as repressor of VirB. Taken together, it seems very likely that
99 ChvH fine-tunes the levels of vir regulon components for adequate stress response, which is comparable to the Cad system.
In the U.S., approximately 9.4 million people contract foodborne diseases each year, resulting in over 1,300 deaths, of which 11% derive from Salmonella enterica (Mead et al., 1999; Scallan et al., 2011). These bacteria are able to overcome the host immune system and to multiply in varieties of mammalian cells while residing in so-called Salmonella-containing vacuoles (SCV) (Bakowski et al., 2008; Prost et al., 2007). Via horizontal gene transfer,
S. enterica acquired several pathogenicity islands, which are essential for host invasion and survival in the new intracellular environment (Haraga et al., 2008). Crucial for Salmonella
virulence are two type III secretion systems, which are encoded on the pathogenicity islands 1 and 2 (SP-1 and SP-2) (Hansen-Wester & Hensel, 2001). Consistent with the chvH deletion in
A. tumefaciens, deletions of yjeK or yjeA lead to attenuation in virulence and altered gene expression in S. enterica, including genes found on SPI-1 (Navarre et al., 2010). The discovery of the role of EF-P in translation now opens up the possibility to determine proteins, which protein levels are very likely affected in these mutants. Several important virulence proteins contain polyproline-stretches, such as the type III secretion ATPase SsaN and the secretion system enhancer protein SseF, both of which are located on SPI-2. Moreover, EnvZ and PhoR, which regulate the transcription of SPI-1 and SPI-2 (Ellermeier & Slauch, 2008; Garmendia et al., 2003), also contain polyproline-stretches. Furthermore, Zou
et al. found out that the overexpression of the poorly characterized porin KdgM is the reason for the susceptibility to antibiotics, hypoosmotic stress and several detergents in efp mutants observed by Navarre et al. (Navarre et al., 2010; Zou et al., 2012). In the plant pathogen
Erwinia chrysanthemi, KdgM is important for the transport of long oligogalacturonides derived from pectines (Blot et al., 2002). Interestingly, kdgM expression is repressed by OmpR, a transcriptional activator that needs to be phosphorylated by the histidine kinase EnvZ for activity (Cai & Inouye, 2002; Condemine & Ghazi, 2007; Egger & Inouye, 1997). As mentioned above, EnvZ contains three consecutive prolines making it a promising target for EF-P.
Genomic analyses reveal an abundance of polyproline-stretches not only in bacteria, but also in archaea and eukaryotes. For example, ~1,000 (13%) yeast proteins contain at least three consecutive proline residues, whereas over 6,000 (20%) polyproline-containing proteins are present in humans. eIF5A has been shown to be involved in several human diseases. For example, Ruhl et al. described an interaction between eIF5A and the viral protein Rev, which is needed for HIV-1 mRNA transport out of the nucleus into the cytoplasm (Ruhl et al.,
100 1993). Thus it was discussed, if eIF5A also plays a role in mRNA transport. However, Rev contains a cluster of three consecutive prolines, thereby leading to the suggestion that eIF5A is rather important for the proper translation of Rev than for mRNA transport.
Furthermore, high levels of eIF5A could be detected in cancer cells making it an appropriate target as tumor marker (Balabanov et al., 2007; Lee et al., 2010; Preukschas et al., 2012). High amounts of mutated p53 can be found in malign cells as well. Normally, the transcription regulator p53 leads to cell cycle arrest under genotoxic stress (Pellegata et al., 1996). During this arrest, the cells have time to repair the DNA or to provoke apoptosis. Unregulated overproduction of p53 however leads to reduced cell cycle arrest, thus cells are able to divide themselves uncontrolled leading to the formation of tumors. Interestingly, p53 is a very proline rich protein containing a cluster of three consecutive prolines, but also several clusters of two prolines including the sequence PPG. eIF5A seems to mediate the translation of p53, thus contributing to a high level of mutated p53 in tumor cells.
Taken together, it could be demonstrated that EF-P and eIF5A play a major role for plant and mammalian diseases. Blocking of eIF5A/EF-P or of hypusination/lysinylation via pharmacological substrates will be of great importance for further studies in respect to find new antibiotics and therapies against bacterial infections, HIV and cancer (Balabanov et al., 2007; Doerfel et al., 2013; Hauber et al., 2005; Lee et al., 2010).
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