Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus.

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSau, with RelSau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.

The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus.

Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S aureus and found that the LAC*ΔgdpP strain of S aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-ß-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.

Cross-talk between two nucleotide-signaling pathways in Staphylococcus aureus.

Nucleotide-signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to external stimuli. The stringent response alarmones guanosine tetra- (ppGpp) and pentaphosphate (pppGpp) control a global response allowing cells to adapt to starvation conditions such as amino acid depletion. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that this signaling nucleotide is essential for the growth of Staphylococcus aureus, and its increased production during late growth phases indicates that c-di-AMP controls processes that are important for the survival of cells in stationary phase. By examining the transcriptional profile of cells with high levels of c-di-AMP, we reveal a significant overlap with a stringent response transcription signature. Examination of the intracellular nucleotide levels under stress conditions provides further evidence that high levels of c-di-AMP lead to an activation of the stringent response through a RelA/SpoT homologue (RSH) enzyme-dependent increase in the (p)ppGpp levels. This activation is shown to be indirect as c-di-AMP does not interact directly with the RSH protein. Our data extend this interconnection further by showing that the S. aureus c-di-AMP phosphodiesterase enzyme GdpP is inhibited in a dose-dependent manner by ppGpp, which itself is not a substrate for this enzyme. Altogether, these findings add a new layer of complexity to our understanding of nucleotide signaling in bacteria as they highlight intricate interconnections between different nucleotide-signaling networks.

Cyclic di-AMP: another second messenger enters the fray.

Nucleotide signalling molecules contribute to the regulation of cellular pathways in all forms of life. In recent years, the discovery of new signalling molecules in bacteria and archaea, as well as the elucidation of the pathways they regulate, has brought insights into signalling mechanisms not only in bacterial and archaeal cells but also in eukaryotic host cells. Here, we provide an overview of the synthesis and regulation of cyclic di-AMP (c-di-AMP), one of the latest cyclic nucleotide second messengers to be discovered in bacteria. We also discuss the currently known receptor proteins and pathways that are directly or indirectly controlled by c-di-AMP, the domain structure of the enzymes involved in its production and degradation, and the recognition of c-di-AMP by the eukaryotic host.

Systematic identification of conserved bacterial c-di-AMP receptor proteins.

Nucleotide signaling molecules are important messengers in key pathways that allow cellular responses to changing environments. Canonical secondary signaling molecules act through specific receptor proteins by direct binding to alter their activity. Cyclic diadenosine monophosphate (c-di-AMP) is an essential signaling molecule in bacteria that has only recently been discovered. Here we report on the identification of four Staphylococcus aureus c-di-AMP receptor proteins that are also widely distributed among other bacteria. Using an affinity pull-down assay we identified the potassium transporter-gating component KtrA as a c-di-AMP receptor protein, and it was further shown that this protein, together with c-di-AMP, enables S. aureus to grow in low potassium conditions. We defined the c-di-AMP binding activity within KtrA to the RCK_C (regulator of conductance of K(+)) domain. This domain is also found in a second S. aureus protein, a predicted cation/proton antiporter, CpaA, which as we show here also directly binds c-di-AMP. Because RCK_C domains are found in proteinaceous channels, transporters, and antiporters from all kingdoms of life, these findings have broad implications for the regulation of different pathways through nucleotide-dependent signaling. Using a genome-wide nucleotide protein interaction screen we further identified the histidine kinase protein KdpD that in many bacteria is also involved in the regulation of potassium transport and a PII-like signal transduction protein, which we renamed PstA, as c-di-AMP binding proteins. With the identification of these widely distributed c-di-AMP receptor proteins we link the c-di-AMP signaling network to a central metabolic process in bacteria.

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress.

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

Surface proteins that promote adherence of Staphylococcus aureus to human desquamated nasal epithelial cells.

BACKGROUND: The natural habitat of Staphylococcus aureus is the moist squamous epithelium in the anterior nares. About 20% of the human population carry S. aureus permanently in their noses and another 60% of individuals are intermittent carriers. The ability of S. aureus to colonize the nasal epithelium is in part due to expression of surface proteins clumping factor B (ClfB) and the iron-regulated surface determinant A (IsdA), which promote adhesion to desquamated epithelial cells present in the anterior part of the nasal vestibule. S. aureus strain Newman defective in IsdA and ClfB exhibited reduced but not completely defective adherence to squamous cells in indicating that other cell surface components might also contribute. RESULTS: Surface proteins IsdA, ClfB, and the serine-aspartic acid repeat proteins SdrC, SdrD and SdrE were investigated to determine their contribution to the adherence of S. aureus to desquamated nasal epithelial cells. This was achieved by expression of ClfB, IsdA, SdrC, SdrD and SdrE on the surface of the surrogate Gram-positive host Lactococcus lactis and by isolating mutants of S. aureus Newman defective in one or more factor. The level of adherence of strains to squamous cells isolated from the nares of volunteers was measured. Results consistently showed that ClfB, IsdA, SdrC and SdrD each contributed to the ability of S. aureus to adhere to squamous cells. A mutant lacking all four proteins was completely defective in adherence. CONCLUSION: The ability of S. aureus Newman to adhere to desquamated nasal epithelial cells is multifactorial and involves SdrD and SdrC as well as ClfB and IsdA.