Two novel point mutations in clinical Staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection

Abstract

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3', 5'-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens.

Figure 1. Clinical features and antimicrobial therapy.

The major features of the clinical case and the relevant clinical isolates are demonstrated. Included are photos of overnight cultures on HBA depicting the normal MRSA strain (JKD6210) and the SCV strain (JKD6229) isolated after many weeks of failed antimicrobial therapy. Pulsed field gel electrophoresis results of SmaI digested DNA from the paired clinical isolates are also included (lane 1 JKD6210, lane 2 JKD6229) and demonstrate an identical banding pattern for the two strains. #Note, JKD6229 demonstrated reduced linezolid susceptibility within the susceptible MIC range.

Figure 2. Microarray transcriptional analysis of SCV strain JKD6229 and parental strain JKD6210.

A) Results of microarray transcriptional analysis of JKD6229 (SCV) compared to JKD6210 (MRSA). Up-regulated genes (in red) are differentially up-regulated in JKD6229 compared to the parent strain JKD6210, and the down-regulated genes (in green) are differentially down-regulated in JKD6229 compared to JKD6210. The heat map analysis highlights the proportion of each cluster of orthologous groups (COG) functional group that is differentially regulated in the array analysis. This clearly demonstrates global transcriptional changes in the SCV strain, affecting genes from all COG groups. J is associated with translation, ribosomal structure and biogenesis; K is related to transcription; L is related to replication, recombination and repair; B is related to chromatin structure and dynamics; D is related to cell cycle control, cell division, chromosome partitioning; V is related to defence mechanisms, T is related to signal transduction mechanism; M is related to cell wall, membrane and envelope biogenesis; N is related to cell motility; U is related to intracellular trafficking, secretion, and vesicular transport; O is related to posttranslational modification, protein turnover, chaperones; C is related to energy production and conversion; G is related to carbohydrate transport and metabolism; E is related to amino acid transport and metabolism; F is related to nucleotide transport and metabolism; H is related to coenzyme transport and metabolism; I is related to lipid transport and metabolism; P is related to inorganic ion transport and metabolism; Q is related to secondary metabolites biosynthesis, transport and catabolism; R and S are function unknown or general function prediction only categories. B) Anti-capsule type 5 immunoblot of serial dilutions of crude capsule extracts from JKD6210, JKD6229, and control strains Newman (Cap5 positive) and P1 (Cap8 positive), demonstrating a marked increase in capsule production in the SCV strain JKD6229, consistent with the microarray transcriptional profiles.

Figure 3. Sequence comparison of pJKD6210 and pUSA300-HOU-MS.

Linear comparison (Artemis Comparison Tool) of the ∼15 kb plasmid detected in S. aureus JKD6210 (pJKD6210) compared with pUSA300-HOU-MS . Replication and regulatory genes are colored red, recombination/transposition genes are blue, antibiotic/heavy metal/bacteriocin resistance genes are brown, hypothetical genes are colored orange. Blue vertical bars indicate the regions of pJKD6210 and pUSA300-HOU-MS sharing high DNA sequence identity. Note; blaZ, beta-lactamase; blaR, beta-lactamase regulator; bin, invertase; sin, recombinase; repA, replication protein.

Figure 4. Location of RelA mutation in JKD6229 and impact of the mutation on cellular ppGpp levels.

A) Alignment of the N-terminal Rsh domains of RelA/SpoT from S. mutans (Rsh_SM) and RelA from S. aureus JKD6210 (Rsh_SA). The triangles are regions shown by Hogg et al. that-when mutated-affect hydrolase function. Indicated by star and grey shading is the F128Y amino acid substitution that occurs in SCV JKD6229 (Rsh_SA_SCV). B and C) Analysis of ppGpp levels in JKD6210 (MRSA), JKD6229 (SCV) and JKD6301 (JKD6210 with relA F128Y mutation) using the fluorescent chemosensor PyDPA . B) Five, two-fold serial dilutions (1/2–1/32) of test strains demonstrate increased ppGpp levels by increased fluorescence in JKD6229 and JKD6301 compared with the parental strain JKD6210. Control 1 is JKD6210 exposed to serine hydroxymate; control 2 is JKD6210 without the addition of PyDPA; control 3 is buffer alone with PyDPA added. (C) Results confirmed in a 96-well plate format, analysed with a fluorescent plate reader. Results are presented as the mean±SD of biological replicates with a significant increase in fluorescence found for JKD6229 and JKD6301 compared to the parental strain JKD6210 (P<0.0001).

Figure 5. Phenotypic features and cellular attachment, invasion and persistence of clinical isolates and mutant strain JKD6301.

A and B) Growth characteristics of parental strain JKD6210 (normal MRSA), the clinical SCV strain (JKD6229), and the allelic exchange relA mutant containing the F128Y mutation (JKD6301). Reduced growth rate in MH broth (A) and reduced colony size on HBA agar (B) is demonstrated for the SCV strain JKD6229. The relA mutant JKD6310 demonstrates reduced growth rate in broth and on solid media, but is not as impaired as the clinical SCV strain. The growth curves were significantly different for all strains (P<0.001). An analysis of cellular attachment/invasion after 1 hour incubation demonstrates a reduced rate of attachment in JKD6229 and JKD6301 compared to JKD6210 (C), however an analysis of cellular invasion after 2 hours incubation (D), demonstrates significantly greater invasion for the SCV strain JKD6229 compared to JKD6210 and JKD6301. Results are presented as mean±SD of triplicates from at least three independent experiments. **, P≤0.01; ***, P≤0.001.

Figure 6. Galleria mellonella virulence assay.

The percent survival of caterpillars injected with the clinical isolate pair (JKD6210, normal MRSA; JKD6229, SCV) and the relA mutant JKD6301 over 96 hours post injection is demonstrated using a Kaplan Meier plot. The average initial inoculum per caterpillar and the colony counts from selected worms after 48 hours incubation are also demonstrated. The difference in survival between JKD6210 and JKD6229 or JKD6301 was significant (P<0.0001), and the difference between JKD6229 and JKD6301 was also significant (P = 0.02). Note: cp = caterpillar.