After subcutaneous (S/C) injection of the +NLL or –NLL strains together with 25 µL of 25 mM Anl, we excised and homogenized whole abscesses after 4, 12, and 16 h of labeling. Importantly, the size of the lesion formed and the number of colony-forming units (CFUs) obtained from each lesion did not depend on the strain used or on the presence of Anl, suggesting that BONCAT does not interfere with the progress of the infection (Fig. 3.2) B). The 530 proteins detected in all three environments were then compared using LIMMA and confirmed that NLL-MetRS was expressed at similar levels between the replicates (Fig. S3.6A), highlighting the constitutive nature of the selected promoter .
Log-fold changes of proteins detected during skin infection were compared with cells grown aerobically or anaerobically, and a Mann–Whitney U test was performed to determine whether the median values for proteins with a given annotation were higher or lower than those without an annotation. Five genes involved in iron acquisition from heme (IsdA, IsdB, IsdC, IsdG and IsdI) were up-regulated during infection (Figure 3.3D, Figure S3.9), which is important due to the low availability of iron in the mammalian host ( 25). Proteins upregulated in the arginine catabolic mobile element (ACME: ArcC2 and ArcB), which are important for resistance to the acidic environment of the skin (26), and in the endogenous arginine catabolic pathway (ArgF and ArcC1), which degrade arginine under anaerobic conditions (Fig. 3.3D, Figure S3.10).
Therefore, we performed principal component analysis (PCA) on the proteins found in all samples; the repeats were clustered in three distinct subspaces (Figure 3.3E, Figure S3.16). Performing PCA on the proteins found in the aerobically and anaerobically grown samples revealed a single component that accounted for 71% of the variance between the data sets. Projecting data from skin infection replicates onto this dimension formed a distinct group (Figure S3.5D).
To determine whether each protein contributed specifically to skin infection, we infected mice using the same skin infection model we used to obtain our proteomic results and monitored the size of the lesion formed. On the fifth day of infection, we excised and homogenized each abscess and made serial dilutions to count the number of CFUs present. Of the mutants tested, only one demonstrated a decrease in the severity of infection compared to wild-type MRSA in both the size of the lesion formed (Fig 3.4B, Fig S3.6) and the number of CFUs within the abscess (Fig 3.4C, S3.6).
Virulence of the ΔadhE mutant was restored in both lesion size (Fig 3.4D) and number of CFUs recovered (Fig 3.4E) by this type of complementation, suggesting that indeed, this protein was contributing to virulence. Furthermore, it was one of the main proteins that PCA revealed contributed to differences in expression profiles between in vitro and in vivo samples (Fig S3.16). We sought to determine whether the catalytic activity of the AdhE protein contributed to infection.
Entamoeba histolytica, the alcohol dehydrogenase activity of the C-terminal domain was dependent on the presence of an active N-terminal aldehyde dehydrogenase domain (38). The C258A mutation reduced infection levels to those of ΔadhE, indicating that a mutation within a single residue of AdhE is enough to reduce virulence (Figs. 3.5D, 3.5E, S3.8). We also did not observe differences in growth rate in the presence of disulfiram (Fig S3.13).
Injection of disulfiram into mice when they were infected with MRSA led to a significant reduction in both the size of the lesion and the number of recovered CFUs compared to vehicle (Fig. 3.5E, 3.5F).
Discussion
This portrait of MRSA infection reveals a pathogen that hides from the innate immune system while foraging for nutrients by expressing proteins important for host cell lysis, antibody evasion, iron acquisition, and amino acid catabolism (Figure 3.3). While many of these proteins have previously been found to be important for infection, we also identified some that have not previously been implicated in contributing to infection. We hypothesize that many of these could be candidates for anti-infective targets, but have not had the ability to screen every protein found.
By examining ten proteins significantly regulated or uniquely found within the mouse compared to in vitro growth, we hoped to find proteins that are important for MRSA infection but not essential for normal growth and viability. Targeted inhibitors of these proteins may impose less selective pressure on the microbe and suppress the development of antibiotic resistance. Disruption of AdhE does not cause growth defects in vitro, which may have led to it not previously being identified as important for MRSA infection (Fig S3.11).
For example, we also found that a related alcohol dehydrogenase (Adh) was highly upregulated in the skin infection proteome, but when we tested a Δadh mutant in vivo, we saw no decrease in its ability to infect (Fig 3.4A-C) . To understand the molecular mechanism of AdhE's contributions to infection and fermentation, the abscess will require further study. The redox sensor Rex regulates expression of AdhE, suggesting that during adaptation to early infection, S.
Previous studies have shown that the pathogenic nature of MRSA depends on its ability to ferment within the low-oxygen environment of the abscess. We hypothesize that more specific therapeutics related to the structure of disulfiram can be designed to target the C258 residue in S. Overall, targeting AdhE and pathogenic fermentation in general has therapeutic potential as it enhances the innate immune system and decreases the severity of infection.
Finally, we hypothesize that application of this approach to other pathogens and animal models of infection would be straightforward and could provide important insights into both the dynamic environment that pathogens encounter during infection and their subsequent response to these fluctuations.
Materials & Methods
Using the same method, the empty plasmid pLL102 was also introduced into wild-type JE2 and the ΔAdhE strain for controls. Samples were brought to 1% SDS and the manufacturer's instructions were followed for the Click-It kit with alkyne-TAMRA (Thermo Scientific). After washing, samples were blocked with 1% mouse serum, then treated with a 1:3000 dilution of anti-Staphylococcus aureus antibody (polyclonal, rabbit) (Ab37644, Abcam).
Insoluble proteins were removed and the reverse lysate was reduced for 10 min in 10 mM dithiothreitol (DTT), followed by incubation with 100 mM iodoacetamide. An equivalent volume of 8 M urea was added, followed by 30 μL of washed DBCO-agarose, and the samples were rotated bottom-up in the dark for 16–24 h. Mice received 160 mg/kg disulfiram (pharmaceutical grade, Sigma) or vehicle the day before and on the day of infection.
2017) Mouse model of hematogenous implant-related Staphylococcus aureus biofilm infection reveals therapeutic targets. 2010) Targeting alpha-hemolysin by active or passive immunization decreases the severity of USA300 skin infection in a mouse model. Bateman BT, Donegan NP, Jarry TM, Palma M, & Cheung AL (2001) Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its use to demonstrate the role of sigB in microcolony formation. Paprotka K, Giese B, & Fraunholz MJ (2010) Codon-enhanced fluorescent proteins in the study of Staphylococcus aureus host-pathogen interactions.
Bubeck Wardenburg J, Williams WA, & Missiakas D (2006) Host defense against Staphylococcus aureus infection requires recognition of bacterial lipoproteins. Carneiro CRW, Postol E, Nomizo R, Reis LFL, & Brentani RR (2004) Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus. 2017) Amino acid catabolism in Staphylococcus aureus and the function of carbon catabolic repression. 2016) Resistance to acute killing of macrophages promotes respiratory fitness of common community-acquired Staphylococcus aureus strains.
2003) Heme-iron passage across the envelope of Staphylococcus aureus. 2013) Functional Modularity of the Arginine Catabolic Movement Element Contributes to the Success of Methicillin-Resistant Staphylococcus aureus USA300. 2004) Staphylocloccus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Cascioferro S, Totsika M, & Schillaci D (2014) Sortase A: An ideal target for antivirulence drug development. 2012) A single copy integration vector that integrates into an engineered site on the Staphylococcus aureus chromosome. Fuchs S, Pane-Farre J, Kohler C, Hecker M, & Engelmann S (2007) Anaerobic gene expression in Staphylococcus aureus. 2014) The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7.
Redox sensing by a Rex family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus. Vitko NP, Spahich NA, & Richardson AR (2015) Glycolytic dependence of nitric oxide resistance and high-level virulence in Staphylococcus aureus.
