Molecular genetic analysis of a locus required for resistance to antimicrobial peptides in Salmonella typhimurium.

The innate immunity of vertebrates and invertebrates to microbial infection is mediated in part by small cationic peptides with antimicrobial activity. Successful pathogens have evolved mechanisms to withstand the antibiotic activity of these molecules. We have isolated a set of genes from Salmonella typhimurium which are required for virulence and resistance to the antimicrobial peptides melittin and protamine. Sequence analysis of a 5.7 kb segment from the wild-type plasmid conferring resistance to protamine contained five open reading frames: sapA, sapB, sapC, sapD and sapF, organized in an operon structure and transcribed as a 5.3 kb mRNA. SapD and SapF exhibited similarity with the 'ATP binding cassette' family of transporters including the bacterial Opp and SpoOK, involved in the uptake of oligopeptides; the yeast STE6, necessary for the export of a peptide pheromone; and the mammalian mdr, which mediates resistance to chemotherapeutic agents in cancer cells. SapA showed identity with other periplasmic solute binding proteins involved in peptide transport. The SapABCDF system constitutes a novel transporter for enteric bacteria and the first one harboring a periplasmic component with a role in virulence.     

Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans

Genetic analysis of host-pathogen interactions has been hampered by the lack of genetically tractable models of such interactions. We showed previously that the human opportunistic pathogen Pseudomonas aeruginosa kills Caenorhabditis elegans, that P. aeruginosa and C. elegans genes can be identified that affect this killing, and that most of these P. aeruginosa genes are also important for mammalian pathogenesis. Here, we show that Salmonella typhimurium as well as other Salmonella enterica serovars including S. enteritidis and S. dublin can also kill C. elegans. When C. elegans is placed on a lawn of S. typhimurium, the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days. This killing requires contact with live bacterial cells. The worms die with similar kinetics when placed on a lawn of S. typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E. coli. After the transfer to E. coli, a high titer of S. typhimurium persists in the C. elegans intestinal lumen for the rest of the worms' life. Furthermore, feeding for 5 hours on a 1:1000 mixture of S. typhimurium and E. coli followed by transfer to 100% E. coli, also led to death after several days. This killing correlated with an increase in the titer of S. typhimurium in the C. elegans lumen, which reached 10,000 bacteria per worm. These data indicate that, in contrast to P. aeruginosa, a small inoculum of S. typhimurium can proliferate in the C. elegans intestine and establish a persistent infection. S. typhimurium mutated in the PhoP/PhoQ signal transduction system caused significantly less killing of C. elegans.     

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A

Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrA(C) strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3'-acyloxyacyl-linked myristate (C(14)) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group.     

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.     

Resistance to antimicrobial peptides in Gram-negative bacteria

Antimicrobial peptides (AMPs) are present in virtually all organisms and are an ancient and critical component of innate immunity. In mammals, AMPs are present in phagocytic cells, on body surfaces such as skin and mucosa, and in secretions and body fluids such as sweat, saliva, urine, and breast milk, consistent with their role as part of the first line of defense against a wide range of pathogenic microorganisms including bacteria, viruses, and fungi. AMPs are microbicidal and have also been shown to act as immunomodulators with chemoattractant and signaling activities. During the co-evolution of hosts and bacterial pathogens, bacteria have developed the ability to sense and initiate an adaptive response to AMPs to resist their bactericidal activity. Here, we review the various mechanisms used by Gram-negative bacteria to sense and resist AMP-mediated killing. These mechanisms play an important role in bacterial resistance to host-derived AMPs that are encountered during the course of infection. Bacterial resistance to AMPs should also be taken into consideration in the development and use of AMPs as anti-infective agents, for which there is currently a great deal of academic and commercial interest.     

Resistance of Salmonella typhimurium mutants to galactose death

A class of galactose-resistant mutants has been derived from strains of Salmonella typhimurium which are defective in uridine diphosphoglucose-4-epimerase. Resistant strains are phenotypically similar to parent organisms but do not lyse in the presence of galactose. Low levels of functional epimerase can be detected in induced cells grown at 20 C but not at 37 C, and acid is not produced from galactose. Sufficient galactose is synthesized at reduced temperatures to fabricate smooth lipopolysaccharide and acceptor sites for phage P22 from galactose-deficient media. The leaky nature of these mutants may account for resistance to galactose death by maintaining galactose metabolites at a subcritical level. Glucose protects sensitive strains by control of levels of toxic metabolites by catabolite repression.     

Degradation of proline peptides in peptidase-deficient strains of Salmonella typhimurium.

A mutant strain of Salmonella typhimurium that lacks two proline-specific peptidases (peptidases P and Q) could not complete the degradation of proline peptides formed as intermediates in starvation-induced protein breakdown. The wild-type strain produced free proline as the product of degradation of proline-labeled proteins. The pepP pepQ mutant, however, produced a mixture of small proline peptides. In the absence of peptidase Q only, peptidase P could complete the degradation of most of the proline peptide intermediates formed. In the absence of peptidase P only, about 50% of the proline-labeled, acid-soluble products were proline peptides. These results are consistent with in vitro specificity data indicating that peptidase Q hydrolyzes X-Pro dipeptides only, whereas peptidase P attacks both X-Pro dipeptides and longer peptides with X-Pro at their N-termini. A mutant strain lacking four broad-specificity peptidases (peptidases N, A, B, and D), but containing peptidases P and Q, also produced proline peptides as products of protein breakdown. This observation suggests that broad-specificity peptidases are required to generate the X-Pro substrates of peptidases P and Q. A strain lacking six peptidases (N, A, B, D, P, and Q) was constructed and produced less free proline from protein breakdown than either the pepP pepQ strain or the pepN pepA pepB pepD strain. These observations suggest that the degradation of peptide intermediates involves the sequential removal of N-terminal amino acids and requires both broad-specificity aminopeptidases (peptidases N, A, and B) and the X-Pro-specific aminopeptidase, peptidase P.     

A two-gene balance regulates Salmonella typhimurium tolerance in the nematode Caenorhabditis elegans

Lysozymes are antimicrobial enzymes that perform a critical role in resisting infection in a wide-range of eukaryotes. However, using the nematode Caenorhabditis elegans as a model host we now demonstrate that deletion of the protist type lysozyme LYS-7 renders animals susceptible to killing by the fatal fungal human pathogen Cryptococcus neoformans, but, remarkably, enhances tolerance to the enteric bacteria Salmonella Typhimurium. This trade-off in immunological susceptibility in C. elegans is further mediated by the reciprocal activity of lys-7 and the tyrosine kinase abl-1. Together this implies a greater complexity in C. elegans innate immune function than previously thought.     

PLK-1 asymmetry contributes to asynchronous cell division of C. elegans embryos

Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis elegans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanisms by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL-1/CHK-1 dependent checkpoint in P1, but how the remaining time difference is controlled is not known. Here, we establish that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by A-P polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1, but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, our findings support a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1-dependent preferential retardation in P1 and PLK-1-dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development.     

Effect of interferon type I on the in vivo persistence of Salmonella typhimurium

The influence of type I interferon on the persistence of S. typhimurium in the body of mice has been studied. The injection of the preparation of interferon has been shown to be conductive to the survival of the animals and to reduce the time of Salmonella persistence in the body. The injection of interferon enhances the phagocytic activity of macrophages in the peritoneal exudate of mice.     

Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli.

During bacterial growth, cell wall peptides are released from the murein and reused for the synthesis of new cell wall material. Mutants defective in peptide transport were unable to reutilize cell wall peptides, demonstrating that these peptides are taken up intact into the cytoplasm prior to reincorporation into murein. Furthermore, cell wall peptide recycling was shown to play an important physiological role; peptide transport mutants which were unable to recycle these peptides showed growth defects under appropriate conditions. Using mutants specifically defective in each of the three peptide transport systems, we showed that the uptake of cell wall peptides was mediated solely by the oligopeptide permease (Opp) and that neither the dipeptide permease (Dpp) nor the tripeptide permease (Tpp) played a significant role in this process. Our data indicate that the periplasmic oligopeptide-binding protein has more than one substrate-binding site, each with different though overlapping specificities.