Whole-genome sequencing reveals a link between β-lactam resistance and synthetases of the alarmone (p)ppGpp in Staphylococcus aureus

Abstract

The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to β-lactam antibiotics: in cultures of such strains, the majority of cells display only a low level of methicillin resistance--often close to the MIC breakpoint of susceptible strains. Yet, in the same cultures, subpopulations of bacteria exhibiting very high levels of resistance are also present with variable frequencies, which are characteristic of the particular MRSA lineage. The mechanism of heterogeneous resistance is not understood. We describe here an experimental system for exploring the mechanism of heterogeneous resistance. Copies of the resistance gene mecA cloned into a temperature-sensitive plasmid were introduced into the fully sequenced methicillin-susceptible clinical isolate S. aureus strain 476. Transductants of strain 476 expressed methicillin resistance in a heterogeneous fashion: the great majority of cells showed only low MIC (0.75 μg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 μg/ml) was also present with a frequency of ∼10(-4). The genetic backgrounds of the majority and minority cells were compared by whole-genome sequencing: the only differences detectable were two point mutations in relA of the highly resistant minority population of bacteria. The relA gene codes for the synthesis of (p)ppGpp, an effector of the stringent stress response. Titration of (p)ppGpp showed increased amounts of this effector in the highly resistant cells. Involvement of (p)ppGpp synthesis genes may explain some of the perplexing aspects of β-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.

FIG. 1.

Properties of Staphylococcus aureus strain 476 and its laboratory constructs carrying plasmid-borne copies of the mecA gene. The mecA gene was introduced into strain 476 by transduction using the temperature-sensitive plasmid pSTSW-2C—as described in the “Materials and Methods” section. (A) shows the oxacillin-susceptibility profile of the transductant 476(pmecA) (■) as determined by its population analysis profile (PAP). Also shown are the PAPs of the cultures of the susceptible recipient strain 476 (□); the highly resistant mutant 476^mut(pmecA) (▲), which was obtained by picking a rare colony present in the culture of 476(pmecA), and the PAP of strain 476^mut (Δ), which was generated from 476^mut(pmecA) by deletion of the thermosensitive mecA plasmid. The PAP of 476(pmecA) was also determined by supplementing each oxacillin-containing plate with 0.03 μg/ml mupirocin (•). (B) shows colony sizes of strains 476, 476(pmecA), and 476^mut(pmecA) on TSA plates incubated at 30°C for 48 hr. Also shown are growth rates (mass doubling times) of the same cultures grown in TSB at 30°C.

FIG. 2.

Schematic representation of methods used to generate various derivatives of the transductants of strain 476. 476^mut cells free of the pmecA plasmid were produced by incubation of 476^mut(pmecA) at 42°C, resulting in 95% small colonies (named 476^mut-cured-small) and 5% large colonies (named 476^mut-cured-large). Reintroduction of the pmecA plasmid into 476^mut-cured-small cells produced bacteria with homogenous and high-level resistance to oxacillin indistinguishable from that of the original 476^mut(pmecA) transductants.

FIG. 3.

Structure of the relA gene in Staphylococcus aureus strains 476(pmecA) and location of the two-point mutations in the relA gene of 476^mut(pmecA). In the 736 aa-long wild-type RelA protein, the Pfam search found an aa 57–156 match hydrolase domain (HD; E-score=10⁻¹⁷); an aa 247–357 match synthetase domain (SYN; E-score=10⁻⁵¹); and an aa 402–461 and 661–727 match, respectively, TGS and ACT (respective E-scores=10⁻²⁶ and 10⁻⁷). In the 2,211-bp-long relA gene, the actual base changes are g→a at the 1,052nd base introducing a new premature stop codon and g→c at the 1,546th base. The former substitution caused the mutant RelA protein lack of TGS and ACT domains.

FIG. 4.

Oxacillin resistance and cellular amounts of (p)ppGpp. (A) Cell extracts were obtained from identical amounts of strains 476(pmecA) and 476^mut(pmecA). The relative amounts of (p)ppGpp were determined by thin layer chromatography followed by autoradiography of extracts from ³²P-labeled bacteria. The sample from 476(pmecA)+mupirocin was used as a control for the (p)ppGpp spots on TLC plates. (B) Relative amounts of (p)ppGpp are expressed as a fraction of the total ³²P-labeled guanine phosphate precursor pool.