If you were a bacteria, how would you avoid the immune system and your would-be killer, the macrophage? Neisseria gonorrhoeae, the pathogen that causes gonorrhea (an STI also known as “the Clap”), has developed a revolutionary strategy to address this issue: form a protective bubble and hide among the enemy (macrophages). In the United States alone, over 1.5 million people are infected with gonorrhea annually, according to the Centers for Disease Control (CDC). N. gonorrhoeae is an obligate pathogen, meaning that it needs to infect human cells in order to reproduce. Its status as an obligate pathogen necessitates that it finds a way around the immune system to survive, as we are its only source of infectable cells. Morphologically, N. gonorrhoeae is gram-negative, meaning that it has an inner and outer membrane with a cell wall in between. N. gonorrhoeae appear as diplococci, or pairs of two cocci (circular-shaped bacteria).
Figure 1: Scanning electron micrograph from He et al. showing N. gonorrhoeae using pili to anchor themselves to cells.
N. gonorrhoeae is able to infect a person in a variety of places, with localization most common in the urogenital tract, according to the American Academy of Family Physicians. The urogenital tract includes the bladder, kidney, ureter, urethra, testes, vagina and uterus. The problem is that N. gonorrhoeae doesn’t always show symptoms, and even when it does, they symptoms tend to be nonspecific. The CDC cites symptoms of an infection of the urogenital tract as consisting of: burning when peeing, increased discharge, abnormal bleeding between periods, and painful/swollen testicles. Most of these symptoms can present due to reasons other than gonorrhea, and may only appear once complications have already taken root. As gonorrhea is the second most common STI worldwide, its tendency to evade antibiotics grows increasingly troublesome. Understanding how N. gonorrhoeae infects human cells while avoiding antibiotics is key to the development of future treatments.
Before we begin, we need to understand how N. gonorrhoeae invades our bodies. Infection begins when the bacteria make contact with epithelial (lining) cells. The bacteria use hair-like tendrils called ‘pili’ to anchor themselves to the surface of the epithelial cells before acting like a grappling hook and retracting the pili in order to pull themselves closer to the cell surface until they finally make contact. When enough bacteria aggregate on the surface of the epithelial cells, they form a micro-colony. This signals for the epithelial cells to ‘eat’ the invading bacteria. Some of the bacteria survive this process, and are able to move through the host cells into the submucosal layer, which is the layer between the lining of an organ (the mucosa) and the surrounding muscle. From there, invading bacteria have full access to other cells, nerves, and the bloodstream. However, epithelial cells are not the only cell type present in urogenital tissue. Macrophages (white blood cells that ‘eat’ and kill pathogens) are also present in high concentrations alongside the aforementioned epithelial cells. N. gonorrhoeae’s efficiency in invading the urogenital tract indicates that N. gonorrhoeae is able to evade death by macrophages (also known as phagocytosis).
Figure 2: Illustration of the steps to N. gonorrhoeae infection by Hill et al.; (1) Bacteria use pili to attach to epithelial cells, (2) pili retract to bring bacteria in contact with cell, (3) epithelial cell eats bacteria, (4) bacteria passes through submucosal layer, (5) a sample bacteria having infected an immune cell, (6) bacteria is able to enter bloodstream.
In their 2021 study, Ivanov et al. found that N. gonorrhoeae cells are able to evade the immune system by invading macrophages, hiding from the host immune system. Moreover, they found that while invading macrophages, N. gonorrhoeae cells not only survive but can thrive, comfortably replicating inside macrophages. Similarly to how N. gonorrhoeae invade epithelial cells, N. gonorrhoeae cells colonize the surface of a macrophage before deploying FMNL3, an actin-nucleating factor. Actin-nucleating factors are proteins that speed up actin polymerization (the polymerization of the microfilaments that make up the cell cytoskeleton). Essentially, N. gonorrhoeae cells release FMNL3 on the surface of a macrophage to signal the actin microfilaments that make up the membrane to reshape itself, encircling the surface colony of N. gonorrhoeae. In doing this, a protective bubble is formed around N. gonorrhoeae to reside in while hiding out within the macrophage- safe from other immune cells (who are now unable to detect their presence). Although intracellular N. gonorrhoeae colonization necessitated FMNL3, expression of the Arp2/3 complex (which is used as an actin nucleating factor in phagocytosis) was not used by invading N. gonorrhoeae. Interestingly, the way FMNL3 encircles the surface colony is similar to how the Arp2/3 complex usually would do the same thing- the main difference being that the Arp2/3 complex would do this with the end result being phagocytosis (which would kill the N. gonorrhoeae cells).
Figure 3: Illustration of the pathway N. gonorrhoeae takes to form an intracellular colony within macrophages, adapted from Ivanov et al. Figure 10.
In their paper, Ivanov et al. took several transmission electron micrographs of macrophages (strain U937) infected with N. gonorrhoeae (strain FA 1090) at a 10:1 ratio, observing how N. gonorrhoeae interact with macrophages. In Figures 4B and C below, N. gonorrhoeae cells house themselves within a protective bubble inside macrophages. At the same time, inset 2 illustrates cells residing within the membrane of the macrophage- a rarer alternative survival mechanism.
Ivanov et al. used fluorescence markers to color different components of their micrograph in Figure 4D. Notably, they inoculated the macrophages with a red fluorescence marker called dsRed-mem, which binds to actin in the cell membrane. Using antibodies marked with Alexa fluorescent markers, the scientists highlighted outer N. gonorrhoeae colonies in green and internal N. gonorrhoeae colonies in pink. By using different colors in their micrographs, the scientists were able to distinguish between different parts of the macrophage-bacteria system and quantify these results. The fluorescence micrograph in Figure 4D shows an overlap of actin (red) and intracellular N. gonorrhoeae (pink), indicating that intracellular N. gonorrhoeae are housed within a membranous bubble inside macrophages. Further, the way in which actin surrounds N. gonorrhoeae does not match how actin acts during phagocytosis, which means that the actin polymerization in Figure 4D cannot be accounted for by phagocytosis. Ultimately, if the phenomenon in Figure 4D cannot be attributed to phagocytosis, then it supports the claim that actin filaments found in the membranes of macrophages are what is reshaped to create intracellular niches for N. gonorrhoeae proliferation.
Figure 4: Figure adapted from Ivanov et al. Figure 6, highlighting large and small intracellular N. gonorrhoeae colonies (sections B and C) as well as actin microfilaments surrounding the intracellular colonies (section D).
The ability of N. gonorrhoeae to form a protective bubble within macrophages suggests that N. gonorrhoeae has evolved to use multiple niches to maximize its chances of survival over time. By hiding within macrophages specifically, N. gonorrhoeae has developed a niche in which it is able to avoid detection by immune agents that target extracellular pathogens (like antibodies and even macrophages themselves). Hiding within macrophages clearly aids in the continued survival and proliferation of N. gonorrhoeae by virtue of not being killed off by immune cells outside of the macrophage in which they are hiding. At the same time, staying on the exterior of macrophages has its own benefits- namely, avoiding internal cell responses to infection. Assume the cell realizes a pathogen has infected it. In that case, the cell can target and kill the invading pathogens through processes like autophagy (programmed cell death and recycling) and lysosomal degradation (being digested by lysosomes).
In the future, there is great potential for future studies on the nuances of how N. gonorrhoeae evade our immune system. By further studying the pathogenesis of N. gonorrhoeae, scientists can develop new treatments for gonorrhea. Currently, the standard treatment for uncomplicated cases of gonorrhea is the administration of ceftriaxone, an antibiotic administered as a shot/injection. Alternative antibiotics include azithromycin, gentamicin, and doxycycline. Growing antibiotic resistance combined with a lack of preventative treatments leaves room for the development of new treatments to enter the market. Perhaps by targeting the various niches (-inter and -intra cellular), we can create a future where antibiotic-resistant gonorrhea is no longer a concern.
About the Author:
Abigail Whiting '26 is a Junior at Mount Holyoke College, majoring in biology and minoring in chemistry. Abigail plans to attend dental school, where she hopes to become a pediatric dentist eventually. Abigail is particularly interested in the intersection between microbiology and oral hygiene, focusing her studies at Mount Holyoke on cell and molecular biology. In her spare time, Abigail enjoys digital art and walking her dog.
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