Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice

Abstract

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa) phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

Figure 1. Pulsed field electrophoresis pattern of Sma I digests from the Staphylococcus aureus A170 strain.

The A172 strain (not shown) displayed an identical pattern. M: DNA Size Standard, Lambda Ladder (Concatemers of λ cl857 Sam7) (Bio-Rad Laboratories, Hercules, CA).

Figure 2. Differences between the phage M^Sa-sensitive strain A170 and the phage M^Sa-resistant strain A172.

The strain A172 grows in larger clumps (A) compared to the strain A170 (B). Bacteria were grown in liquid culture and collected for microscopic examination at the early stationary phase. (C) The A172 strains displays also a slower growth rate, compared to A170.

Figure 3. In S. aureus, phage-resistance comes with production of capsular polysaccharide.

(A) Strains A170, A177, A179, A181 (sensitive to the phages M^SA, M^SA1, M^Sa2, M^SA3, respectively). (B) Strains A172, A178, A180, A182 (resistant to the phages M^SA, M^SA1, M^Sa2, M^SA3, respectively).

Figure 4. Expression levels of S. aureus A170 and A172 virulence factors.

Acquisition of phage-resistance by A172 is accompanied by extensive alterations in virulence gene expression levels. Out of the 14 ORFs examined, 13 are significantly under-expressed, compared to the phage-sensitive strain A170. Bacteria were collected for transcriptional analysis during the exponential growth phase. SA0107 (spa, IgG binding protein A precursor); SA0112 (hypothetical protein, similar to cysteine synthase); SA0184 (hypothetical protein); SA039 (geh; glycerol ester hydrolase); SA1007 (α-haemolysin); SA1160 (nuc; thermonuclease); SA1898 (hypothetical protein, similar to SceD precursor); SA2097 (hypothetical protein, similar to secretory antigen precursor SsaA); SA2206 (sbi; IgG-binding protein SBI); SA2255 (oligopeptide transporter substrate binding protein); SA2406 (gbsA; glycine betaine aldehyde dehydrogenase gbsA); SA2459 (ica; N-glycosyltransferase); SAR216 (groEL; chaperonin GROEL); SAR2117 (groES; co-chaperonin GRES). ORFs numbers correspond to the S. aureus N315 genome sequence. Gene designations (when known) and proteins function are shown in parentheses.

Figure 5. Phage M^SA is inhibited by N-acetyl-glucosamine (GlcNAc), the teichoic acid from A170 (A170^TA) or by treatment with N-acetyl-glucosaminidase (GlcNA-ase) from Canavalia ensiformis; phage M^SA is not inhibited by the teichoic acid from A172 (A172^TA) or glucose.

(A) GlcNAc inhibits the lysis of the A170 strain by the phage M^Sa, while glucose (B) does not. (C) A170^TA inhibits the lysis of the A170 strain by the phage M^SA, while A172^TA does not. (D) The phage-sensitive strain A170 grows in the presence of the M^Sa phage, if pre-treated with GlcAc-ase (4 U/tube; 2 h at 37°C).

Figure 6. Gas chromatography-combined mass spectrometry spectra of the teichoic acids from the A170 and A172 strains.

Acetylated O-methyl glycosides from the phage–sensitive A170 and phage-resistant A172 strains of S. aureus. Loss of ribitol (A) and t-GlcNAc linked to ribitol (B) in the teichoic acids is part of the strategy adopted by A172 to gain phage-resistance. The peak of 4-GlcNAc was chosen as reference. Gro: glycerol; GlcNAc: N-acetylglucosamine; ManN: N-acetylmannosamine; MurNAc: N-acetyl muramic acid; Glc: glucose; C15:0: pentadecanoic acid; C16:0: esadecanoic acid; C18:0: octadecanoic acid; C20:0: eicosanoic acid; 4-GlcNAc: 4-linked N-acetylglucosamine; 3GlcAcNMaN: 3-linked N-acetylmannosamine; t-GlcNAc: terminal GlcNAc.

Figure 7. Anti-inflammatory activity of the A172 vaccine.

A172 modulates transcription of the genes coding for pro-inflammatory cytokines (TNF-α, INF-γ and IL-1β) and induces transcription of the genes coding for anti-inflammatory cytokines (Il-4 and Il-6).

Figure 8. Gas liquid chromatography profile of monosaccharides obtained by acid hydrolysis of the capsular polysaccharide from S. aureus A172.

Abundance expresses the relative ratio between monosaccharides.