The question has puzzled scientists for decades – how are L-form bacteria able to divide if they lack cell walls? The answer remained elusive until recently, when medical researcher Josep Casadesus at the University of Sevilla, Spain published on the subject in the medical journal Bioessays.^[1] The findings he reports provide valuable clues about how L-form bacteria are able to propagate and reproduce.

In the case of the more commonly studied classical bacterial forms, the creation of a septum, or a wall separating two cavities or spaces, is an essential requisite for cell division. The main structure of a septum is composed of chains of amino acids made from the substance peptidoglycan. In fact, classical bacterial divide only after a double layer of peptidoglycan is laid down in the middle of the cell. The septum subsequently splits – forming two daughter cells. The septum itself is formed by several different proteins that unite to form a ring-like complex.

Interestingly, according to the standard view of L-form bacteria, the pathogens lack peptidoglycan and thus do not form a septum when they divide. This belief stems from the work of prominent L-form scientist Louis Dienes who, when looking at the pathogens under the electron microscope during the 1930s, saw that, in contrast to classical bacteria, they are not surrounded in peptidoglycan.

But if Dienes’ observation is correct, then how are L-form bacteria able to reproduce? “A major enigma raised by L-forms is how they manage to free cell division of its normal dependence on peptidoglycan synthesis,” states Casadesus. “This question has in fact puzzled the experts on cell division over the decades.”

It turns out that the classical definition of L-forms as consistently cell-wall-less bacterial variants may need a revision in order to accommodate recent observations by Richard d’Ari and coworkers at the Institut Jacques Monod in Paris.^[2]

The team recently published the results of a study demonstrating that in order for classical forms of bacteria to be converted to the L-form – a feat usually accomplished by exposing the classical forms to one of the beta-lactam antibiotics – some components of a cell wall are indeed formed at the time of division.

The researchers used the beta-lactam antibiotic cefsulodin to convert classical cells of E. coli into its L-form. They proceeded to investigate which (if any) of the known E. coli cell division functions were required for L-form propagation. But how? The team grew L-form mutants, each without a specific enzyme needed to create a different protein that makes up the E. coli septum used for division. These proteins included D-glutamate, diaminopimelic acid, and muramic acid. Interestingly, when the synthesis of each of these cell wall proteins was blocked, the mutant L-forms were unable to divide or propagate.

After performing further biochemical analyses on his L-forms, D’Ari found that the cefsulodin-induced L-forms do actually contain peptidoglycan – about 7% of what their classical counterparts would harbor. This amount of peptidoglycan is far too small to allow them to form cell walls that would span their entire circumference, but the researchers believe it is just enough peptidoglycan to allow for the creation of septums when the L-forms need to divide.

The bottom line: L-form bacteria may indeed form small, temporary cell walls when they divide. “L-forms retain the ability to synthesize small amounts of peptidoglycan and this residual synthesis is essential for their propagation,” states Casadesus. Whether D’Ari’s findings will apply to all species of L-form bacteria remains to be seen.

Interestingly, Casadesus pauses frequently throughout his discussion of the bacterial cell wall to discuss how L-form bacteria are likely to be the pathogens responsible for causing many illnesses of “unknown cause.” “Of special interest for human health is the formation of L-forms as a consequence of specific antibiotic treatments, and the potential involvement of L-forms in persistent and relapsing infections,” states the scientist.

In fact, Casadesus seems quite convinced that L-form bacteria are responsible for causing a wide array of chronic diseases. One can’t help but wonder if part of this author’s impetus for writing was to, once again, remind the medical community of the key role that L-form bacteria play in causing disease.

According to Casadesus, the idea that L-forms might underlie chronic and relapsing bacterial infections is supported by:

1. studies which show that the pathogens are spared from the killing actions of antibodies which act only on bacteria that possess complete cell walls.

2. The fact that the pathogens can persist inside white blood cells of the immune system called phagocytes and perhaps emerge in more virulent forms if a patient becomes immunocompromised.

3. The reality that L-forms can survive for long periods of time within the macrophages (white blood cells), explaining “both the difficulty of showing the presence of L-forms in human blood cultures and the failure of certain antibiotic treatments.”

Right on, Dr. Casadesus! Luckily, biomedical researcher Trevor Marshall has created a treatment that can kill L-form bacteria despite the survival mechanisms presented above – meaning that for the first time in history patients are recovering from a wide array of chronic diseases.

Returning to the subject of bacterial cell walls, Casadesus concludes, “Not only has D’Ari’s work provided novel support to the idea that peptidoglycan synthesis is absolutely needed for cell division, but it has also corrected an old, insufficiently grounded dogma.”

Casadesus’ effort to rethink dogma is admirable. Let’s hope it’s the first of many, many more to come that will gradually wear away at the conventional wisdom surrounding so many aspects of chronic disease – finally ushering us into an era that embraces a new understanding of the immune system and the pathogens making us sick.

New insights into culturing L-forms

And yet, D’Ari’s insights into the characteristics of L-form bacteria do not stop at cell wall division. In what is also groundbreaking work, his research team was also able to come up with a new, more effective protocol for culturing L-form bacteria in the first place.

In a review article published in the Journal of Bacteriology that also discusses D’Ari’s L-form research, Dr. Kevin Young at the North Dakota School of Medicine describes how other research teams have, up until this point, spent long hours in the lab generating L-forms by “incubating the cells in complex media in the presence of high concentrations of penicillin, growing them as embedded colonies in a specific percentage of agar, and passaging them multiple times for several years,” D’Ari’s team has broken new ground by discovering a way to culture L-forms using a new advanced technique.^[3]

According to Young, the novel procedure is “extremely straightforward” and centers on incubating the pathogens for only one night in the presence of the antibiotic cefulodin.

D’Ari’s team developed their procedure by studying the substances that play a role in the process of L-form transformation. According to D’Ari, in the lab, classical forms of bacteria transform into the L-form only if they are denied the ability to form a normal cell wall. The beta-lactam antibiotics work towards this end by blocking the creation of proteins known as penicillin-binding proteins (PBPs) – the proteins responsible for forming the cross-linked chains that compose a proper cell wall. When the ability of the PBPs to create a full cell wall is blocked, “the cells become spherical, osmosensitive [sensitive to water], and heterogeneous in size” – in essence they become L-form bacteria.

However, there are several different PBPs, and one of D’Ari’s most important discoveries is that in order to most effectively create L-forms, only some, and not all PBPs must be inactivated at the time of formation. For example, when creating E. coli L-forms, PBPs 1a and 1b should be inactivated, but PBP 3 and PBP 2 should remain active if the pathogens are to grow correctly.

Unfortunately, several of the beta-lactam antibiotics that researchers have used to grow L-forms in the past (including penicillin) inactivate ALL the PBPs. That means that in the future, it will be of extreme importance for L-form researchers to work with an antibiotic (such as cefulodin) that controls the activity of the PBPs in the correct manner. D’Ari’s new procedure “finally achieves a proper balance between the set of reactions (involving PBPs) to be inhibited versus those that must be retained,” states Young.

This is big news. Looking back, it’s not hard to understand why, without these recent insights, researchers trying to create L-forms often used the wrong antibiotics, forcing them to resort to using much more complicated and time-consuming laboratory procedures in order to create the pathogens. One of the major reasons behind why L-form research has often been marginalized is because, over the past century, few scientists have possessed the patience or the knowledge to use such complex culturing techniques. But now that the process of creating L-forms has been streamlined, perhaps more microbiologists will be enticed to study these pathogens in more detail.

The history of L-form research is also haunted by scientists who tried and failed to culture the pathogens, because the laboratory methods used to grow them were so complicated. For the most part, instead of acknowledging possible errors, these researchers chose to explain the failure of their experiments by simply concluding that there were no L-form bacteria in their samples. This caused many researchers to miss the connection between L-form bacteria and a variety of chronic diseases and goes a long way towards explaining why, even today, many doctors and researchers are not even aware that L-form bacteria can exist within the human body.

Now that we know more about the exact methods needed to successfully grow L-forms, errors in cultivation should become much less common, meaning that perhaps awareness of these pathogens and the diseases they cause will also grow.

“This [D’Ari’s work] should place the investigation of L forms on a much firmer foundation and should revive interest in, and reinvigorate the study of, this interesting mode of prokaryotic existence,” states Young.

These findings also sparked my interest for another reason. If it holds true that in order for L-form bacteria to form from classical bacteria, some of the PBPs must be inactivated, could scientists in the future create drugs that work to keep all PBPs active, thus maintaining a stable cell wall that prevents L-form formation? After all, the Marshall Protocol effectively kills L-form bacteria and stops their ability to replicate once they have formed, but the key to truly quelling illness will be to stop these bacteria from being created from classical forms in the first place. Blocking inactivation of the PBPs seems like a tantalizing prospect to slow or stop the formation of L-form bacteria in the first place….at least in my mind! Whether this will actually prove possible remains to be seen.

REFERENCES

1. Casadesús, J. (2007). Bacterial L-forms require peptidoglycan synthesis for cell division.BioEssays : news and reviews in molecular, cellular and developmental biology, 29(12), 1189-91. [↩]
2. Joseleau-Petit, D., Liebart, J., Ayala, J. A., & D’Ari, R. (2007). Unstable Escherichia coli L Forms Revisited: Growth Requires Peptidoglycan Synthesis. J. Bacteriol., 189(18), 6512-6520. [↩]
3. Young, K. D. (2007). Reforming L Forms: They Need Part of a Wall After All? J. Bacteriol., 189(18), 6509-6511. [↩]