Master’s Project

December 25, 2011

In the lab of Prof. A.J.M. Driessen (Molecular Microbiology) I am currently working on the first of my two master’s projects. This project has an approximate duration of six months and is centered on some molecular aspects of bacterial protein secretion across the cytoplasmic membrane. In order to give you the chance of learning something about what I do,  I summarized my work-plan and some of the theory behind it.  If you have questions, do not hesitate to contact me!

Background

Cells are no closed and just internally highly complex entities that are sealed from the outside world. In fact in one way or the other every cell directly or indirectly communicates with its environment through processes which are mediated by channels or receptors. For multicellular eukaryotic organisms it is evident and well-known that cells secrete substances which serve as substrates for a wide array of physiological functions. These substances are ranging from simple ions for neuronal signaling purposes or pH homeostasis to more complex peptide hormones and other proteins. A general issue which was overlooked by science for decades is the fact that secretion and communication also appears in and among prokaryotic cells. Since these organisms most of the time consist of single cells, systemically relevant excretion of compounds which are synthesized within the cell is occurring in a different manner and on a different scale. Bacteria, representing one of the two prokaryotic domains, indeed possess highly complex systems which enable interaction with the environment in the form of other cells or an adherence substrate.

A key role for a bacterial cells ability to survive and to secrete molecules into the environment plays the bacterial secretory (Sec) system. Genetic, biochemical, biophysical, and structural approaches during the last two decades have shaped a detailed understanding of the functional elements and dynamics of this system. The so-called translocase system encompasses an array of proteins which are functionally centered around the translocon channel which mediates the export of proteins across the bacterial cytoplasmic membrane and the insertion of membrane proteins into it. The focus of this work lies on the study the SecA motor proteins that occur in bacteria that next to the general Sec system also possess a so-called accessory Sec system which is responsible for the export of a subset of proteins. A significant number of pathogenic bacteria make use of this subsystem to export protein virulence factors. SecA is a motor protein and ATPase which is thought to be mainly responsible for the movement of proteins across the membrane via the SecYEG translocon channel. How the two different SecA motor proteins that occur in the general and accessory Sec systems interact and what their exact functional relationship is, is currently not understood. By making use of genetic, biochemical, and fluorescence microscopic techniques the focus of the present study is to elucidate a possible interaction between Staphylococcus aureus’ general Sec systems SecA1 and its accessory Sec systems counterpart SecA2. Parts of the bacterial Sec system are conserved in all three kingdoms of life and among others also function in the eukaryotic endoplasmic reticulum in order to secrete proteins into the cytoplasm. Embedding the performed research into its context is essential and therefore brief introductions to bacterial cell structure and the two Sec systems follow with a focus on SecA structure and function.

It does, however, not lie within the scope of this work to formulate a wholesome review on the translocase system. A number of reviews have already been published on this topic, which offer an in-depth overview and cover this subject more explicitly (1, 2, 3).

The Sec translocase system

Since the SecA proteins of S. aureus, which stand central in my research project, are part of the functional complex Sec translocase system, here a brief overview is given about this system. Fig. 1 summarizes the most important aspects of this kind of bacterial protein translocation across the cytoplasmic membrane.

Fig. 1: Schematic overview of the bacterial protein translocation system termed Sec translocase. Proteins which are synthesized within the ribosome are exported from the cytoplasm over the cytoplasmic membrane into the periplasm by the proton motive force (PMF) and the ATPase SecA via the heterotrimeric membrane channel SecYEC. Two major ways of protein translocation exist: posttranslational secretion and cotranslational insertion into the cytoplasmic membrane. Secretory proteins are either directly targeted to SecA by means of their N-terminal signal sequence or are bound by the chaperone SecBfirst and translocate later via SecA and SecYEG. Membrane proteins are translocated cotranslational. Their C-terminal signal sequence is bound by the signal recognition particle (SRP) and targeted to the SRP receptor (FtsY). Consequently SecAtranslocates these proteins via a lateral gate in the SecYEG channel into the membrane. SecDF(yajC) is an accessory factor which seems to improve preprotein translocation. YidC associates with the translocon during protein insertion into the membrane (figure derived from (1)).

Some bacteria posses two different versions of SecA

In bacteria SecA universally functions as the ATPase which delivers the chemical energy to physically translocate proteins across the cytoplasmic membrane in a post-translational manner. However, some bacteria posses two homologues of this protein termed SecA1 and SecA2. SecA1 functions as the essential housekeeping protein for translocation, while SecA2 seems to be especially important for a subset of proteins. Studies on bacteria which are expressing  SecA2 have shown, that this subset of proteins often includes virulence factors, as for example adhesion molecules (4). In addition to SecA1/A2 some of these bacteria also contain a homologue monomer of the heterotrimeric translocon channel SecYEG (Fig. 1) termed SecY2.  Summarizing, bacteria containing either only SecA1/A2 or SecA1/A2 + SecY2 are said to express an accessory Sec system next to the general Sec system which is depicted in Fig. 1.

The question

The intriguing research questions of my project therefore is: Do SecA1 and SecA2 dimerize with each other or which other combinations are required to be functional? This is an important question, since SecA1/A2 might function as a link between the general and accessory Sec systems. As the accessory Sec system is important for virulence in some bacteria it is essential to learn more about its protein molecular basis. Fig. 2 summarizes the hypothetical dimers which SecA1/A2 might form in bacteria with or without an additional SecY2 homologue. Currently I am investigating the SecA proteins of S. aureus which is not expressing an additional SecY2. Some of the combinations depicted below (Fig. 2, (D) through (F)) therefore are not predicted to apply to the current state of my project.

Fig. 2: Schematic overview of the hypothetical combinations of two SecA motor proteins in S. aureus with the two SecYEG homologues when assuming a functional SecAdimer. Dimers (A) through (C) might function with the canonical SecY1EG translocation channel, while dimers (D) through (F) might also interact with the accessory SecY2EG translocation channel which is present in S. aureus. In addition it is also theoretically possible that dimer (A) binds to SecY2EG and dimer (D) to SecY1EG (created by myself, based on earlier work of Irfan Prabudiansyah).

How to get there

Here I will present a very brief work-plan, just to give you an idea about which techniques and experimental approaches my project entails in order to investigate the properties of SecA1/A2 and their functional relation to each other in S. aureus. The aim is to use molecular genetics to overexpress both proteins, which is followed by using knowledge of biochemistry to purify and fluorescently label the proteins. The final step is to use confocal microscopy (cross-correlation) and FRET microscopy to examine the potential interaction between both proteins.

Genetics

The molecular cloning of of both secA1 and secA2 gene sequences from S. aureus into plasmid vectors involves some bioinformatics work and the designing of the needed primers. In addition DNA sequencing is performed to confirm correct sequence insertion. The Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible plasmids are isolated and heat-shock transformed into E. coli cells. These cells containing either secA1 or secA2 are consequently grown in large amounts at 25°C to 30°C to reduce inclusion body formation after overexpression induction by the addition of IPTG into the growth medium. After cell harvesting and cell membrane disruption by french pressing or sonication, the overexpressed protein-containing cytoplasmic fraction is isolated by ultracentrifugation. Results are confirmed by SDS-PAGE and Western blots.

Biochemistry

Overexpressed SecA1/A2 is purified by HPLC mediated cation exchange and size-exclusion chromatography. After desalting on a size-exclusion column both proteins are labeled with two fluorophores (Cy5 & fluoresceine) via a maleimide group to their external cysteines (50% of purified SecA1 is labeled with first fluorophore, other half with second, same procedure for SecA2). Labeling efficiency is checked by photometry at different wavelengths specific to excitations spectra of fluorophores. Removal of excess fluorohore molecules is essential for avoiding too strong background noise during microscope steps. This is achieved by HPLC cation exchange at protein specific salt concentration. Results are checked on SDS-PAGE under fluorescence detection conditions.

Microscopy

Analysis of dimer formation is first achieved by an array of different concentrations of SecA1 and SecA2 combined to each other. Secondly, the potential dimers are analyzed with the help of a confocal microscope observing a volume of approximately 90 femtoliters. Fluorescence cross-correlation spectroscopy is applied to this volume of the solution in order to determine the diffusion coefficient of the fluorescently labeled particles (5). Potential dimer formation can then be described by altered dual color absorption/emission spectra of the proteins (FRET), as well as their unchanged or slower diffusion time through the observed volume in the case of dimer formation.

Final remarks

If you also consider the approach of my work as interesting as I do, you can always contact me back via mail. In the future I would like to continue my work and extend my knowledge into the direction of biophysics. Describing the properties of proteins by interdisciplinary means has definitively caught my eye.

(1) Du Plessis, Nouwen, Driessen, The Sec translocase, Biochimica et BiophysicaActa (2010).

(2) Driessen & Nouwen, Protein Translocation Accross the Bacterial Cytoplsmic Membrane. Annual Reviews of Biochemistry (2008).

(3) Papanikou, Karamanou, Economou, Bacterial protein secretion through the translocase nanomachine, Nature Reviews Microbiology (2007).

(4) Rigel & Braunstein, A new twist on an old pathway – accessory Sec systems, Molecular Microbiology (2008).

(5) Schwille, Mayer-Almes, Rigler, Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution, Biophysical Journal (1997)

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