The Hitchhiker's Guide to the Extracellular Space: Staphylococcus aureus’ Pathway to Potential Pathogenicity via Cytoplasmic Protein Excretion.

Original Article: Ebner, P., Prax, M., Nega, M., Koch, I., Dube, L., Yu, W., Rinker, J., Popella, P., Flötenmeyer, M., Götz, F. (2015). Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus. Molecular Microbiology, 97(4), 775-789.

By Dani Acosta-Saavedra

Background and Significance:

For the longest time researchers believed that, defined transport pathways through pores exclusively drove protein exportation embedded in the membrane of a cell. These proteins contain a signal sequence, which in many way acts as a passport, boarding pass, and GPS all wrapped up into one, allowing the protein in question to localize itself to its exit site and be exported from the cell. However, new research reveals that bacteria have the ability to secrete cytoplasmic proteins (CPs), which lack this signal sequence. Cytoplasmic proteins are proteins that are typically found in and operate in the cytosol of the cell, the gel-like substance that enclosed by the cell membrane.

However, studies that analyzed the composition of the substances secreted from various types of bacteria revealed the presence of different kinds of CPS extracellularly on the cell surface. These observations led researchers to question why the bacteria would choose to secrete proteins with intracellular activity, especially since they lacked the signal sequence that would predestine them for exportation. This gave rise to the idea that CPs are “moonlighting” proteins, which would refute any assumption of the bacteria being wasteful with their resources. The hypothesis is that much like underpaid medical residents who take on second jobs; the bacteria are similarly being economical with their protein production by creating proteins that can serve multiple functions both intracellularly and extracellularly. Talk about efficient!

Gram-stain of S. aureus under a microscope

 shows their cocci shape.

One of the many functions researchers believe CPs play a role in extracellularly is in pathogenesis once a bacterial cell encounters a potential host cell. Understanding the mechanism by which these CPs without signal sequences are released to do their damage is very important to identify possible steps that can be inhibited as a way to disarm the bacteria’s virulence. The possible hypotheses about the CP exportation mechanism remain hotly contended. One side of the debate postulates that the secretion is unplanned to the type of CPs that end up excreted, proposing that they are exported when the cell lyses or bursts open. This hypothesis would suggest excretion happens during cell death. The opposing hypothesis argues that not only is CP excretion protein selective, it is also time specific and exit site specific. Evidence that supports this hypothesis comes from secretome studies in which researchers observed that only a specific set of CPs were found in the supernatant, indicating that these excretions did not occur at random. However, proposing a mechanism of export is dependent on evidence that suggests a selective mode of excretion rather than indiscriminate secretion, which is exactly what the authors of this paper set out to do.

Summary

The authors studied the excretion patterns of two CPs with glycolytic enzyme activity, aldolase and enolase in the aerobic Staphylococcus aureus, both which have been found outside the bacteria. S. aureus is a gram-positive bacterium that is spherically shaped. Although it can be part of the friendly and necessary microflora in humans, it has a tendency of going over to the dark side and becoming a pathogen. It is most known for causing staph infections in hospitals and athletic locker rooms in the form of MRSA, which is a type of S. aureus with the super-villain power of antibiotic resistance. In addition, the experimenters ran experiments on the IgG binding protein Sbi, which is a factor that allows the pathogen to suppress the host's immune system by anchoring itself to the cell’s surface (Smith et al 2011). Once present it can bind to immunoglobulins, which are antibodies produced by the body that tag the pathogen as a means of alerting the body’s law enforcement cells to mount a response. Much like a smooth talking driver that goes over the speed limit, S. aureus avoids getting a “ticket” and therefore can disguise itself, preventing engulfment by macrophages, the immune system’s SWAT team. The authors chose Sbi because of its role in virulence and its already well-defined export mechanism to be able to compare the excretion of CPs to (Ebner et al 2015).

To determine if enolase and aldolase secretion was time specific, the experimenters first established the growth curve of the mutant strain of S. aureus, upon which they could then map relative maximum secretion levels for the three proteins, aldolase, enolase, and Sbi against the times on the growth curves. They measured protein accumulation with Western blots, which relies on specific amino-acid sequences in the peptide. Presence of the protein is marked by the appearance of a band in the gel and relative abundance of the protein is indicated by how dark the band is. S. aureus underwent exponential growth between hours 3 and 8. The Western blot analysis for enolase showed the appearance of the darkest bands at hours 3 and 4. The Western blot analysis for aldolase showed the appearance of the darkest bands at hours 6-8. These results indicate that the majority of the CP secretion occurs during exponential phase, during which the cell is growing. This evidence suggests that excretion of CPs is timing specific, which further invalidates the lysis-dependent hypothesis. For both proteins, the relative intensity of the bands decreased once the cell entered stationary phase. However, in both CPs the bands intensity increased once more at 24 hours, which indicates that the later secretion is most likely occurring during cell death. Both cells resembled the secretion pattern of the Sbi protein, exhibiting a bell shaped excretion pattern.

            

A. The researchers monitored the growth curve of S. aureus

mutant strain. B. Measured the amount of CP protein over time.

Darker band signify more protein present. C&D. Graph of tnhe amount

of protein detected against time for the three CPs, enolase,

adolase and sbl.

Now that the researchers could pinpoint the general timing of CP secretion in S. aureus, their next task was to localize the exit site. The first experiment they conducted consisted of tagging the CPs in question with protein specific antibodies that allowed their position to be visualized with fluorescence. Unfortunately, this first experiment failed since they were unable to detect any fluorescence. The researchers reasoned that diffusion from the exit site during normal secretion did not allow the CPs to be concentrated enough to enable tagging. In order to trap the CPs at their exit site the experimenters engineered constructs that fused the CPs with a LysM-binding motif, which exhibits a binding function to the peptidoglycan that forms part of the cell wall. This method ensured that the CPs remained fixed to the cell wall, fixed in time, to allow for fluorescent tagging. The experimenters detected fluorescence concentration exclusively in dividing cells along the septum. In contrast, the CPs could not be visualized 

in intact single cells, suggesting that recruitment of the CPs to their exit sites and secrete them only occurred during cell replication. Choosing to excrete CPs to act as potential toxins when the bacteria are increasing their numbers allows the pathogen to almost multitask and save energy as it spreads in the human host. To further confirm the septum localization, the experimenters visualized the CPs via TEM by tagging the CPs with a primary antibody, which itself was tagged with an immunogold secondary antibody. This method of visualization is visually superior to fluorescent tagging since the gold particles has a higher electron density and are considered “heavier”, which increases the intensity of the image’s contrast. By doing this the researchers ensured that the fluorescent tags observed in the previous experiments were not just artifacts, but rather indicative of the actual CP localization at the septum.

Depiction of secondary antibody tagging.

 Conclusions:

The Sec-pathway of protein secretion,
similar to S. aureus uses to excrete sbl

For now, all the research can suggest is that secretion is indeed selective. However, researchers still need to investigate the underlying mechanism that explains how the septum exit site exactly recruits enolase and aldolase. The similarities in export profile of enolase and aldolase to Sbi, which follows a Sec dependent transport pathway, might suggest that enolase and aldolase hitchhike their way on Sbi’s ride, much like Arthur Dent
in The Hitchhiker's Guide to the Galaxy. Further, experiments to test this hypothesis would involve inhibiting different steps in the Sbi Sec pathway and see if enolase and/or aldolase export is similarly inhibited. Another area of further research would investigate whether this hitchhiking behavior is exclusive to enolase and aldolase collaborating with Sbi, or if these CPs can adapt their behavior to catch “rides” depending on what other well-defined transport proteins are in their vicinity.

Virulence or the harmful characteristics in a microorganism that can cause disease are an important topic of concern. The more we can understand how pathogens evade the immune system and how they attack our healthy cells, the more we can develop strategies to nip their weaponry in the bud.

References

Ebner, P., Prax, M., Nega, M., Koch, I., Dube, L., Yu, W., Rinker, J., Popella, P., Flötenmeyer, M., Götz, F. (2015). Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus. Molecular Microbiology, 97(4), 775-789.

Smith, E.J.M, Visai, L., Kerrigan, S.W., Speziale, P., Foster, T.J. (2011). The Sbi Protein Is a Multifunctional Immune Evasion Factor of Staphylococcus aureus. Infection and Immunity, 79(9), 3801-3809.

Kaiser, G.E. (2009). Gram Stain of Staphylococcus aureus. [photograph]. http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/gpstaph.html

Strauch, E.M., and Surdulescu R. (2004). The general secretion machinery in E. coli. [figure].             http://www.sonic.net/~surdules/projects/XINViewer/report/analysis.html