One of the biggest digital heists happened in February 2025 when Bybit, a Dubai-based cryptocurrency platform, was infiltrated by hackers. After invading the system, the hackers secretly altered the final destination of the crypto coins and successfully reallocated coins worth about 1.5 billion dollars into an unknown account. Like these hackers, who utilize sophisticated methods to avoid cybersecurity tools, some bacteria are also able to disguise themselves as a means of survival following host invasion.
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Say hello to the stealthy infiltrator, Legionella pneumophila which is a gram-negative bacterium that causes a severe form of pneumonia called Legionnaires’ Disease (LD). According to the Centers for Disease Control (CDC), though LD can be treated using antibiotics, approximately 10% of those who contract it will die from the infection. Thus, it comes as no surprise that this pathogen is able to persist in biofilms, protozoa and in eukaryotes.
In humans, infection occurs through the inhalation of contaminated water droplets. Once L. pneumophila gains entry, it colonizes the lungs and replicates in monocytes and macrophages. These immune cells act as sentinels, and eliminate bacteria using intrinsic properties like the digestive enzymes found in their lysosomes.
The life cycle of Legionella pneumophila in a typical eukaryotic cell. Following phagocytosis by a macrophage, L. pneumophila releases effector proteins which allow it to maintain its LCV. In doing so, the bacterium is able to replicate and avoid disruption by digestive enzymes in lysosomes. Source
Since successful host hijacking is dependent upon Legionella’s ability to avoid degradation, the bacterium encases itself in a specialized compartment called the Legionella-Containing Vacuole (LCV). Thua, to develop novel drugs that effectively target Legionella, it is necessary to understand the mechanisms that this pathogen employs to undermine the immune system.
The Icm/Dot type IV secretion system (T4SS) is a prerequisite for the formation of L. pneumophila’s protective cloak. This “molecular needle” translocates loads of effector proteins that work to form and maintain the LCV structure. Among the hundreds of proteins that are transported into the cytoplasm, the Sde family of proteins (SdeA, SdeB, and SdeC) is of particular interest. This is due to their ability to modify other host proteins through ubiquitination—a process whereby a protein is tagged with ubiquitin to control its function.
In the context of L. pneumophila infection, the Sde proteins modify a host protein called reticulon 4 (Rtn4). Rtn4 is heavily involved in controlling the morphology of the Endoplasmic Reticulum (ER). Through the phosphoribosyl-linked ubiquitin transferase activity, which is just an elaborate way of describing how Sde tags other host proteins, the bacterium reorganizes the ER and directs it to surround LCV. By doing so, it provides an enclosement for the bacterium to safely grow and replicate.
While LCV protection is key for L. pneumophila’s survival, prior studies have shown that in the absence of the Sde family of proteins, Legionella still possesses the ability to thrive in mice-derived macrophages, but not in amoeba. Daunting right? For this reason, Kim and Isberg (2023), two researchers from Tufts, sought to uncover alternative proteins or pathways that may offset the loss of function of the Sde proteins in this paper.
First, they used transposon sequencing to identify genes that might be important for Legionella’s replication in macrophages. This technique harnesses a transposon, or a length of DNA that randomly inserts itself to different parts of a genome, to create gene mutations. By sequencing the DNA of surviving bacteria and comparing it to those that don’t, they identified that three bacterial effector genes (sdhA, ridL and legA3) were involved in Legionella’s survival when Sde proteins were absent. The SdhA protein binds to a phosphatase enzyme (OCRL phosphatase) to prevent cell sorting compartments from interacting with the LCV. On the other hand, the RidL protein targets a host cell complex (the retromer) which is involved in intracellular sorting and recycling. Finally, LegA3 is a protein that facilitates Legionella’s replication in several hosts. However, it has yet to be studied with regard to the bacterial replication within the LCV.
Now having identified the aforementioned genes, the researchers used a luciferase reporter assay to confirm the function of the different genes. The assay measured fluorescent signals to quantify the survival of L. pneumophila with consideration to the simultaneous loss of one or more genes encoding the wildtype (WT), Sde, SdhA and DotA proteins. The results are summarized in the figure below.
Figure 2 from Kim and Isberg (2023) measures the quantity of viable L. pneumophila for the different strains over time. The Relative Light Units (RLU) quantifies the bacterial strains by measuring the light produced, which is proportional to the number of bacteria growing inside the macrophages.
Figure 2A shows that the combined loss of ridL and sde (ΔridL/Δsde) results in significant bacterial growth issues as compared to the individual sde (∆sde) or ridL (ΔridL) strains. Moreover, the individual loss ridL (ΔridL) is not enough to hinder the growth of Legionella as illustrated by the overlapping lines for the ΔridL and WT lines on the first graph. Now Figure 2B shows that the combined removal of sde and legA3 (Δsde/ΔlegA3) significantly disturbs the growth of L. pneumophila as compared to the individual WT, Δsde, ΔsdhA, and ΔlegA3 strains. Finally, Figure 2C shows that the combined loss of sdhA and sde (ΔsdhA/Δsde) results in decreased bacterial growth as compared to Δsde, yet its effects on bacterial growth are similar to the dotA- strain. This is unsurprising since the dotA- strain of L. pneumophila is without the canonical Dot/Icm secretion system, typically used for delivering effector proteins into the host cell.
Building upon their findings about the significance of SdhA, Kim and Isberg (2023) sought to determine whether individual Sde effectors (SdeA, SdeB, or SdeC) could compensate for the loss of sdhA, and restore the LCV’s integrity. To answer this question, they reintroduced plasmids encoding single Sde protein effectors to the mutant lacking both sdhA and sde. They stained the macrophages, infected with L. pneumophila, to differentiate between bacteria in the cytosol and bacteria in the LCV. Then, they calculated the percentage of cytosolic bacteria to assess the degree of LVC damage and identify the proportion of bacteria that were at risk for degradation. Their findings are summarized in Figure 4A.
Figure 4A from Kim and Isberg (2023) sheds light on the vacuole integrity of L. pneumophila strains at 2 hours post-infection upon reintroduction of plasmids for the different protein effector genes
The results from Figure 4A demonstrate that reintroducing Sde effectors restores vacuole integrity by significantly reducing the exposure of bacteria to the host cytosol. Thereby underscoring their critical role in stabilizing the Legionella-containing vacuole (LCV).
Overall, the study reveals how L. pneumophila exploits host mechanisms within host cells. It delineates the significance of compartmentalization in the pathogenesis of L. pneumophila and the clever yet self-serving mechanisms employed for survival. These findings underscore how Sde modifies the host ER-associated protein Rtn4 through the synergistic action of other proteins synthesized by Legionella. This allows scientists to develop schematic models, like the one in Figure 7, which can help inform future studies about Legionalla. Since this is just the tip of the iceberg, these results provide an exciting avenue for developing other therapies for other ER-targeting pathogens like Legionella. Could L. pneumophila, or other bacteria, be secretly reengineering other host cell components to stay one step ahead in the cellular heist?
Figure 7 from Kim and Isberg (2023) is a schematic summary of the different processes and proteins involved in the maintenance of the LCV to avoid vacuole disruption.
About the Author:
Akosua Frimpong ‘25 is a Biochemistry major on the pre-med track at Mount Holyoke College. She works in the Lijek lab at Mount Holyoke College to understand how immunopathology proceeds in the female genital tract after Chlamydia infection. She has a deep interest in immunology, cancer biology, and the gut microbiome. She hopes to utilize her knowledge to further advancements in the field of medicine. In her free time, she enjoys going on long walks, spending time with family and trying good food!
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