Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages

Klumpp, J.; Fuchs, Thilo M.

doi:10.1099/mic.0.

Cited by 52 publications

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“…1B) (36). The poor lipid bilayer permeation of the highly polar dithionite species limits the quenching of the NBD moiety to the extracellular space of the cell (37). We reasoned that complete quenching of the fluorescence signal following treatment with sodium dithionite would be consistent with extracellular incorporation of the NBD moiety (Fig.…”

Section: Resultsmentioning

confidence: 99%

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Fura

Kearns

Pires

2015

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Fura

Kearns

Pires

2015

Journal of Biological Chemistry

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“…However, for a given level of X, a statistically significant difference in levels of MH5 α-helicity was observed as pH was varied ( Figure 4B), decreasing from circa 50 % at pH 6 to circa 40 % at pH 8 [F = 98.376; p = 0.001]. In relation to physiological levels of the lipid in membranes of S. aureus, which are typically X = 60 at pH 6, and X = 40 at pH 7 29,[39][40][41][42] , MH5 possessed levels of α-helicity of circa 45 % at pH 7 (X = 40), which increased by around 5 % at pH 6 (X = 60) ( Figure 4B). These results indicate that the levels of α-helicity possessed by MH5 were enhanced by low pH conditions with varying Lys-DOPG having limited influence on α-helicity.…”

Section: Secondary Structure Of Mh5 In the Presence Of Lipid Mimics Omentioning

confidence: 99%

“…This effect in enhanced by low pH conditions, when the production of Lys-PG is upregulated and where levels of the Lys-PG as high as 80 % of total membrane lipid have been reported for the organism 29,[39][40][41][42] .…”

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confidence: 99%

Low pH Enhances the Action of Maximin H5 against Staphylococcus aureus and Helps Mediate Lysylated Phosphatidylglycerol-Induced Resistance

et al. 2016

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Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an improved ability to penetrate (ΔΠ = 6.2 mN m–1) and lyse (lysis = 48%) Staphylococcus aureus membrane mimics, which incorporated physiological levels of lysylated phosphatidylglycerol (Lys-PG, 60%), compared to that at pH 7 (ΔΠ = 5.6 mN m–1 and lysis = 40% at pH 7) where levels of Lys-PG are lower (40%). The peptide therefore appears to have optimal function at pH levels known to be optimal for the organism’s growth. MH5 killed S. aureus (minimum inhibitory concentration of 90 μM) via membranolytic mechanisms that involved the stabilization of α-helical structure (approximately 45–50%) and showed similarities to the “Carpet” mechanism based on its ability to increase the rigidity (C s –1 = 109.94 mN m–1) and thermodynamic stability (ΔG mix = −3.0) of physiologically relevant S. aureus membrane mimics at pH 6. On the basis of theoretical analysis, this mechanism might involve the use of a tilted peptide structure, and efficacy was noted to vary inversely with the Lys-PG content of S. aureus membrane mimics for each pH studied (R 2 ∼ 0.97), which led to the suggestion that under biologically relevant conditions, low pH helps mediate Lys-PG-induced resistance in S. aureus to MH5 antibacterial action. The peptide showed a lack of hemolytic activity (<2% hemolysis) and merits further investigation as a potential template for development as an antistaphylococcal agent in medically and biotechnically relevant areas.

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“…The identity of each spot was determined by reference to 178 the migration pattern of purified phospholipid standards as described previously 49,50 . Each spot was 179 scraped from the plate using a sterile scalpel and the quantity of phospholipid in each determined by 180 digestion with 0.3 ml 70% perchloric acid at 150 o C for 3 h. The digested phospholipid was then 181 incubated with a detection reagent (10% ascorbic acid, 2.5% ammonium molybdate, 5% perchloric 182 acid at 1:1:8 ratio by volume) at 37 o C for 2 h, and quantification by measurement at 750 nm and 183 reference to standards of known concentrations 49,50 . The concentration of each lipid was derived 184 from quantification of lipid in culture supernatant from USA300 WT or ΔagrA mutant using FM-4-64 185 and reference to a standard plot generated using purified phosphatidylglcyerol.…”

Section: Lipid Extraction and Thin-layer Chromatography 157mentioning

confidence: 99%

“…The identity and relative proportions of lipids were determined as described previously 24,49 . Lipids 173 were extracted from spent culture supernatant into chloroform:methanol (2:1 v/v) as described 174 above.…”

Section: Lipid Extraction and Thin-layer Chromatography 157mentioning

confidence: 99%

Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids

et al. 2016

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Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-34resistant Staphylococcus aureus (MRSA) 1,2 . Although resistance is rare, treatment failure can occur 35 in >20% of cases 3,4 and so there is a pressing need to identify and mitigate factors that contribute 36 to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which 37 frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. 38Wild-type S. aureus was killed rapidly by daptomycin but Agr-defective mutants survived 39 antibiotic exposure by releasing membrane phospholipid, which bound and inactivated the 40 antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-41 triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented 42 antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was 43 inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and 44 enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that 45 protects against daptomycin, which can be compromised by Agr-triggered toxin production or an 46 existing therapeutic antibiotic. 47 S. aureus encodes multiple virulence factors, many of which are controlled by Agr 5,6 , a 48 quorum-sensing system encoded by a 4 gene operon (agrBDCA) and a gene encoding a regulatory 49 RNA (RNAIII). However, invasive S. aureus infections often give rise to Agr-defective mutants, 50 typically involving agrA or agrC, hypothesised to provide a selective advantage in the presence of 51 antibiotics [7][8][9][10][11][12][13][14] . To test this hypothesis, we determined the killing kinetics of wild-type S. aureus or agr 52 mutants by clinically-relevant antibiotics. 53Agr status did not affect the rate of staphylococcal killing by vancomycin, oxacillin or 54 gentamicin ( Supplementary Fig. 1, 2). By contrast, whilst wild-type S. aureus was killed by 55 daptomycin, loss of quorum-sensing components of Agr (AgrA or AgrC) enabled S. aureus strains 56 USA300 or SH1000 to survive in the presence of daptomycin during the first 8 hours of exposure (Fig. 57 1a,b). A mutant lacking the regulatory RNAIII component of agr was killed as efficiently as the wild-58 type (Fig. 1a), as were agrA or agrC mutants complemented with the relevant genes on plasmids 59 3 ( Supplementary Fig. 3). After the initial period of killing, CFU counts of both wild-type and agr-60 mutant S. aureus recovered to similar levels by 24 h, without the acquisition of resistance, explaining 61 why all strains had identical daptomycin MIC and MBC values (Fig. 1c, Supplementary Supplementary Fig. 4). This biphasic killing and subsequent recovery profile is similar to several 63 previously reported daptomycin killing assays, although the contribution of Agr to this phenomenon 64 was unknown [15][16][17] . 65In addition to agr-deletion mutants, clinical isolat...

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Identification of novel genes in genomic islands that contribute to Salmonella typhimurium replication in macrophages

Cited by 52 publications

References 0 publications

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Low pH Enhances the Action of Maximin H5 against Staphylococcus aureus and Helps Mediate Lysylated Phosphatidylglycerol-Induced Resistance

Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids

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